Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Patent
1997-08-05
1999-02-09
Saucier, Sandra E.
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
435146, C12N 120, C12P 742
Patent
active
058693190
DESCRIPTION:
BRIEF SUMMARY
This application is a 371 of PCT/JP96/00182 filed Jan. 31, 1996.
FIELD OF THE INVENTION
The present invention relates to a microorganism which is capable of converting bile acids having a 7.alpha.-hydroxyl group or a 7-keto group into bile acids having a 7.beta.-hydroxyl group such as 3.alpha., 7.beta.-dihydroxy-5.beta.-cholanic acid (referred to hereinafter as "ursodeoxycholic acid") which is useful as a cholagogue and a method for producing bile acids having a 7.beta.-hydroxyl group utilizing said microorganism.
BACKGROUND ART
As a chemical production method for converting a 7.alpha.-hydroxyl group of bile acids into a 7.beta.-hydroxyl group, there is known a method in which a 7.alpha.-hydroxyl group is oxidized into a 7-keto group which is then reduced to a 7.beta.-hydroxyl group.
As a microorganism which is capable of converting a 7.alpha.-hydroxyl group of bile acids into a 7.beta.-hydroxyl group, there are known Clostridium absonum (U.S. Pat. No. 4,303,754 and Journal of Lipid Research, 22, 458-465 (1981)) and Clostridium limosum (Journal of Lipid Research, 25, 1084-1089 (1984)). In addition, as a microorganism which is capable of converting a 7-keto group of bile acids into a 7.beta.-hydroxyl group, there are known Peptostoreptococcus productus isolated as an enterobacterium (Acta Med. Univ. Kagoshima., 24 (1), 31-8 (1982)), Eubacterium aerofaciens (Acta Med. Univ. Kagoshima., 24 (1), 43-7 (1982)), and Ruminococcus sp. PO1-3 (J. Biochem. Toyama Medical and Pharmaceutical University, 102, 613-619 (1987)).
However, when a chemical production method is used to oxidize a 7.alpha.-hydroxyl group into a 7-keto group, some of the hydroxyl groups (for example, 3.alpha.-hydroxyl group) other than the 7.alpha.-hydroxyl group are simultaneously oxidized. Those hydroxyl groups, therefore, need to be protected in such cases. Furthermore, in the reduction reaction of a 7-keto group into a 7.beta.-hydroxyl group, the use of metal sodium is common and therefore the method has a risk of explosion. The method, therefore, has some efforts to resolve in terms of handling and safety.
In the method for producing bile acids having a 7.beta.-hydroxyl group by means of a microorganism, the organisms used such as Clostridium absonum, Clostridium limosum, Peptostoreptococcus productu, Eubacterium aerofaciens, and Ruminococcus sp. can grow only at low concentrations of bile acids and besides the conversion efficiency of the bile acid is low, too. Therefore, it was impossible to manufacture bile acids on a commercial scale. For example, the substrate concentrations at which Clostridium absonum and Clostridium limosum convert 3.alpha., 7.alpha., 12.alpha.-trihydroxy-5.beta.-cholanic acid (referred to hereinafter as "cholic acid") into 3.alpha., 7.beta., 12.alpha.-trihydroxy-5.beta.-cholanic acid (referred to hereinafter as "ursocholic acid") at a conversion efficiency of 60% or greater are 0.06% (w/v) and 0.4% (w/v), respectively, and those at which the above organisms convert 3.alpha., 7.alpha.-dihydroxy-5.beta.-cholanic acid (referred to hereinafter as "chenodeoxycholic acid") into ursodeoxycholic acid at a conversion efficiency of 60% or greater are 0.02% (w/v) and 0.04% (w/v), respectively (Journal of Lipid Research, 22, 458-465 (1981); Journal of Lipid Research, 25, 1084-1089 (1984)). In addition, these organisms are strict anaerobes and hence cannot grow in the presence of oxygen. Substitution with nitrogen, therefore, is required at the time of culturing them in order to avoid contact with the air or to expel oxygen, thus necessitating special handling and equipment.
Furthermore, since Clostridium absonum has a pathogenically of causing gas gangrene symptoms, though weak, in the infected humans (Microbiol. Immunol., 23 (7), 685-687 (1979)), care must be taken in its handling in the commercial production. The pathogenic nature is largely due to the hemolysis-causing and lecithinase-producing ability (Journal of Lipid Research, 22, 458-465 (1981), and Infection and Immunity, 9, 15-19 (1974)). Thus, Clostridium limosum
REFERENCES:
patent: 4303754 (1981-12-01), Macdonald
Bergey's Manual of Determinative Bacteriology. Ninth Edition. 1994. pp. 559-562.
Bergey's Manual of Determinative Bacteriology. Eight Edition. 1974. pp. 557-559.
J.D. Sutherland, et al., "The Enzymic and Chemical Synthesis of Ursodeoxycholic and Chenodeoxy From Cholic Acid", Preparative Biochemistry, 12(4) pp. 307-321, 1982.
I.A. Macdonald, et al., "Formation of urso-and ursodeoxy-cholic acids from primary bile acids by Clostridium absonum", Journal of Lipid Research, vol. 22, pp. 458-466, 1981.
J.D. Sutherland, et al., "Formation of urso-and ursodeoxy-cholic acids from primary bile acids by a Clostridium limosum soil isolate", Journal of Lipid Research, vol. 25, pp. 1084-1089, 1984.
Noriyuki Masuda, et al., "A Mass Spectrometrical study of the Epimerization of the 7-Hydroxy Group in Primary Bile Acids by the Combined Action of 7.alpha.- and 7.beta.- Dehydrogenating Organisms", Acta Med. Univ. Kagoshima, 24 (1), pp. 31-38, 1982.
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Taiko Akao, et al., "Purification and Characterization of 7.beta.-Hydroxysteroid Dehydrogenase from Ruminococcus sp. Of Human Intestine", J. Biochem. vol. 103, No. 3, pp. 613-619, 1987.
Shinichi Nakamura, et al., "Isolation of Clostridium absonsum from a Case of Gas Gangrene", Microbiol. Immunol., vol. 23 (7), pp. 685-687, 1979.
M. Hayase, et al., "Isolation of Clostridium absonum and Its Cultural and Biochemical Properties", Infection and Immunity, vol. 9, No. 1, pp. 15-19, Jan. 1974.
R.E. Buchanan, et al., "Bergey's Manual of Determinative Bacteriology", Eighth Edition, 1974, pp. 557-559.
Kimura Hiromi
Ogata To-oru
Okamura Akio
Afremova Vera
Saucier Sandra E.
Tokyo Tanabe Company Limited
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