Title:
新規12α-ヒドロキシステロイドデヒドロゲナーゼ、その製造および使用
Document Type and Number:
Japanese Patent JP2011526481
Kind Code:
A
Abstract:
12alpha -Hydroxysteroid dehydrogenase (12alpha -HSDH), obtained from Clostridium sp.and having a molecular weight of 27-30 kD, is new. Independent claims are included for: (1) 12alpha -HSDH-mutant with a modified cosubstrate-utilization; (2) a nucleic acid sequence comprising (a) a coding nucleic acid sequence that encodes a protein having (a1) a fully defined 270 amino acid (SEQ ID NO. 2) sequence given in the specification or a fully defined 257 amino acid (SEQ ID NO. 4) sequence given in the specification, each beginning at position +1 or +2 and a protein having (a2) an amino acid sequence derived from a sequence according to (a1) with a percentage identity of at least 60%, (b) a coding nucleic acid sequence according to SEQ ID NO. 1 (comprising a fully defined 813 base pair sequence given in the specification) or SEQ ID NO. 3 (comprising a fully defined 774 base pair sequence given in the specification), or (c) a coding nucleic acid sequence derived from the nucleic acid sequence according to SEQ ID NO. 1 or 3 with a percentage identity of at least 60%; (3) an expression cassette comprising the nucleic acid sequence under the genetic control of at least one regulative nucleic acid sequence; (4) a vector comprising at least one expression cassette; (5) recombinant microorganisms that carry at least one nucleic acid sequence or at least one expression cassette, transformed with at least one vector; (6) the preparation of 12alpha -HSDH; (7) a method for enzymatic oxidation of 12alpha -hydroxysteroid, comprising converting the hydroxysteroid in the presence of the above 12alpha -HSDH and optionally isolating at least one of the formed oxidation product from the reaction mixture; (8) a method for enzymatic reduction of 12-ketosteroid, comprising converting the ketosteroid in the presence of the above 12alpha -HSDH and optionally isolating at least one of the formed reduction product from the reaction mixture; (9) a bioreactor comprising 12alpha -HSDH in immobilized form; (10) a method for qualitative or quantitative measurement of 12-ketosteroid and/or 12alpha -hydroxysteroid, comprising subjecting the steroids to a redox reaction that is catalyzed by the 12alpha -HSDH in the presence of redox equivalent, measuring the change in the concentration of the redox equivalent and qualitatively or quantitatively measuring the 12-ketosteroid and/or 12alpha -hydroxy steroid; and (11) a method for preparing ursodeoxy cholic acid compounds of formula (I), comprising (aa1) oxidizing a cholic acid compound of formula (II), in the presence of 12alpha -HSDH, to its corresponding 12-keto chenodeoxycholic acid of formula (III), (aa2) subsequently converting (III), by deoxygenation, to chenodeoxycholic acid of formula (IV), (aa3) chemically oxidizing (IV), in position 7, to 7-keto-lithocholic acid of formula (V), (aa4) reducing (V) and (aa5) optionally further purifying the reaction product. R : alkyl, NR 1>R 2>, H, alkali metal ion or N(R 3>) 4 +>; R 1>, R 2> : not defined; R 3> : H or alkyl; and R 1> 1> : H or acyl. [Image] [Image] [Image].
Inventors:
Aigner, Arno
Gross, Ralph
Schmidt, Rolf
Brown, michael
Mauer, Steffen
Gross, Ralph
Schmidt, Rolf
Brown, michael
Mauer, Steffen
Application Number:
JP2011501147A
Publication Date:
October 13, 2011
Filing Date:
March 25, 2009
Export Citation:
Assignee:
Pharmazel, Gamebecher
International Classes:
C12N15/09; C12N1/15; C12N1/19; C12N1/21; C12N5/10; C12N9/04; C12P33/02; C12P33/06; C12Q1/32
Other References:
JPN6013057474; Ｅｕｒ． Ｊ． Ｂｉｏｃｈｅｍ． Ｖｏｌ．１９６, 1991, ｐｐ．４３９-４５０
JPN6013057475; ＢＩＯＴＥＣＨＮＯＬＯＧＹ ＡＮＤ ＢＩＯＥＮＧＩＮＥＥＲＩＮＧ Ｖｏｌ．２６, 1984, ｐｐ．５６０-５６３
JPN6013057476; ＰＲＥＰＡＲＡＴＩＶＥ ＢＩＯＣＨＥＭＩＳＴＲＹ Ｖｏｌ．１２， Ｎｏ．４, 1982, ｐｐ．３０７-３２１
JPN6013057475; ＢＩＯＴＥＣＨＮＯＬＯＧＹ ＡＮＤ ＢＩＯＥＮＧＩＮＥＥＲＩＮＧ Ｖｏｌ．２６, 1984, ｐｐ．５６０-５６３
JPN6013057476; ＰＲＥＰＡＲＡＴＩＶＥ ＢＩＯＣＨＥＭＩＳＴＲＹ Ｖｏｌ．１２， Ｎｏ．４, 1982, ｐｐ．３０７-３２１
Attorney, Agent or Firm:
Akaoka Michio
Kazuo Akaoka
Kazuo Akaoka