NOVEL 7beta-HYDROXYSTEROID DEHYDROGENASES AND THEIR USE

Abstract

The invention relates to novel 7β-hydroxysteroid dehydrogenases which are obtainable from bacteria of the genus Collinsella , especially of the strain Collinsella aerofaciens , to the sequences encoding said enzymes, to methods for producing said enzymes and to their use in the enzymatic conversion of cholic acid compounds, and especially in the production of ursodeoxycholic acid (UDCS). The invention also relates to novel methods for the synthesis UDCS.

1 . A 7β-hydroxysteroid dehydrogenase (7β-HSDH) obtainable from a bacterium, in particular of the genus  Collinsella , such as the strain  Collinsella aerofaciens  DSM 3979 (ATCC 25986) and functional equivalents derived from the 7β-HSDH, with a molecular weight, determined by gel filtration, of about 53 to 60 kDa. 
     
     
         2 . The 7β-HSDH or functional equivalent derived therefrom as claimed in  claim 1 , which catalyzes
 a) the stereospecific reduction of a 7-ketosteroid to the corresponding 7β-hydroxysteroid, and/or 
 b) the regiospecific hydrogenation of a ketosteroid in the 7-position to the corresponding 7β-hydroxysteroid. 
 
     
     
         3 . A 7β-HSDH comprising an amino acid sequence according to SEQ ID No.: 2 or a sequence derived therefrom with an identity degree of at least 80% to this sequence, with a molecular weight, determined by gel filtration, of about 53 to 60 kDa and which in particular at least catalyzes the stereospecific reduction of a 7-ketosteroid to the corresponding 7β-hydroxysteroid. 
     
     
         4 . A method for the enzymatic synthesis of 7β-hydroxysteroids, wherein the corresponding 7-ketosteroid is converted in the presence of a 7β-HSDH according to the definition in  claim 1 , and at least one reduction product formed is optionally isolated from the reaction system. 
     
     
         5 . The method as claimed in  claim 4 , wherein the ketosteroid to be reduced is selected from dehydrocholic acid (DHCA), 7-keto-lithocholic acid (7-keto-LCA), 7,12-diketo-lithocholic acid (7,12-diketo-LCA) and the derivatives thereof, such as in particular a salt, amide or alkyl ester of the acid. 
     
     
         6 . The method as claimed in  claim 4 , wherein the reduction takes place in the presence (and with consumption) of NAD(P)H. 
     
     
         7 . A method for the enzymatic oxidation of 7β-hydroxysteroids, wherein the hydroxysteroid is converted in the presence of a 7β-hydroxysteroid dehydrogenase according to the definition in  claim 1 , and an oxidation product formed is optionally isolated from the reaction system. 
     
     
         8 . The method as claimed in  claim 7 , wherein the 7β-hydroxysteroid is 3,12-diketo-7β-CA or a derivative thereof, such as in particular a salt, amide or alkyl ester. 
     
     
         9 . The method as claimed in  claim 7 , wherein the oxidation takes place in the presence (and with consumption) of NAD(P) + . 
     
     
         10 . The method as claimed in  claim 6 , wherein the redox equivalents consumed are electrochemically or enzymatically regenerated. 
     
     
         11 . The method as claimed in  claim 10 , wherein consumed NAD(P)H is regenerated by coupling with an NAD(P)H-regenerating enzyme selected from an NAD(P)H dehydrogenase and in particular an alcohol dehydrogenase (ADH). 
     
     
         12 . The method as claimed in  claim 11 , wherein the NAD(P)H-regenerating enzyme is selected from natural or recombinant, isolated or enriched
 a) alcohol dehydrogenases (EC 1.1.1.2) and   b) functional equivalents derived therefrom   
     
     
         13 . A method for the production of ursodesoxycholic acid (UDCA) of the formula (1) 
       
         
           
           
               
               
           
         
         wherein 
         R stands for alkyl, NR 1 R 2 , H, an alkali metal ion or N(R 3 ) 4   + , wherein the residues R 3  are the same or different and stand for H or alkyl, 
         wherein 
         a) a cholic acid (CA) of the formula (2) 
       
       
         
           
           
               
               
           
         
         wherein R has the aforesaid meanings, is optionally chemically oxidized to the dehydrocholic acid (DHCA) of the formula (3) 
       
       
         
           
           
               
               
           
         
         wherein R has the aforesaid meanings; 
         b) DHCA is reduced in the presence of at least one 7β-HSDH according to the definition in  claim 1  to the 3,12-diketo-7β-cholanic acid (3,12-Diketo-7β-CA) of the formula (4) 
       
       
         
           
           
               
               
           
         
         c) 3,12-diketo-7β-CS is reduced in the presence of at least one 3α-hydroxysteroid dehydrogenase (3α-HSDH) to the corresponding 12-keto-ursodesoxycholic acid (12-keto UDCA) of the formula (5) 
       
       
         
           
           
               
               
           
         
         wherein R has the aforesaid meanings, and then 
         d) 12-keto-UDCA of the formula (5) is chemically reduced to UDCA; and 
         e) the reaction product is optionally further purified. 
       
     
     
         14 . The method as claimed in  claim 13 , wherein the steps b) and/or c) are coupled with an (in particular enzymatic) cofactor regeneration step. 
     
     
         15 . The method as claimed in  claim 14 , wherein step b) is coupled with a cofactor regeneration step, in which NADPH is regenerated by alcohol dehydrogenase (ADH) with consumption of a sacrificial alcohol. 
     
     
         16 . The method as claimed in  claim 14 , wherein step c) is coupled with a cofactor regeneration step, in which NADH is regenerated by formate dehydrogenase (FDH) with consumption of formate, or in which NADPH is regenerated by alcohol dehydrogenase (ADH).