Microbial process for preparing pravastatin

Abstract

The present invention relates to a new microbial process for the preparation of the compound formula (I) from a compound of general formula (II) wherein R stands for an alkali metal or ammonium ion, by the submerged cultivation of a mold strain able to 6β-hydroxylate a compound of the Formula (II) in aerobic fermentation and by the separation and purification of the product of Formula (I) formed in the course of the bioconversion. The process comprises cultivating a strain of Mortierella maculata filamentous mold species that is able to 6β-hydroxylate a compound of the general Formula (II), on a nutrient medium containing assimilable carbon and nitrogen sources and mineral salts and separating the product formed from the fermentation broth, then isolating the compound of formula (I) and purifying the same. Novel strains of Mortierella maculata are also disclosed.

1 . A microbial process for the preparation of the compound of formula (I)  
     
       
         
         
             
             
         
       
     
     from a substrate compound of formula (II),  
     
       
         
         
             
             
         
       
     
     wherein R stands for an alkali metal or ammonium ion,  
     comprising the steps of 
 a) cultivating a strain of  Mortierella maculata  filamentous mold species able to 6β-hydroxylate a compound of formula (II) on a nutrient medium containing assimilable carbon and nitrogen sources and mineral salts,  
 b) feeding the substrate to be transformed into the developed culture of  Mortierella maculata,    
 c) fermenting the substrate until the end of bioconversion,  
 d) separating the compound of formula (I) from the culture broth, and  
 e) isolating the compound of formula (I).  
 
   
   
       2 . The process of  claim 1 , wherein said medium is a nutrient broth.  
   
   
       3 . The process of  claim 2 , wherein said step of separating the compound of formula (I) from the culture broth is performed by adsorption on an anionic ion exchange resin.  
   
   
       4 . The process of  claim 2 , wherein said step of separating the compound of formula (I) from the culture broth is performed by extraction with a water immiscible organic solvent, followed by the preparation of its lactone derivative or its secondary amine salt as an intermediate.  
   
   
       5 . The process of  claim 2 , wherein said step of separating the compound of formula (I) from the culture broth is performed by purification of the alkaline aqueous extract obtained from the organic solvent extract of the fermentation broth with chromatography on a non-ionic adsorbing resin.  
   
   
       6 . The process of  claim 1 , wherein the strain of  Mortierella maculata  is the  Mortierella maculata  n. sp. E-97 strain deposited at the National Collection of Agricultural and Industrial Microorganisms, Budapest, Hungary under accession number NCAIM(P)F 001266.  
   
   
       7 . The process of  claim 1 , wherein the strain of  Mortierella maculata  is the  Mortierella maculata  n. sp. E-97/15/13 strain deposited at the National Collection of Agricultural and Industrial Microorganisms, Budapest, Hungary under accession number NCAIM(P)F 001267.  
   
   
       8 . The process of  claim 1 , wherein the hydroxylase enzyme of the strain used for the transformation is induced by 8-de(2-methyl-butyryl) compactin or compactin.  
   
   
       9 . The process of  claim 2 , wherein a compound of formula (II) as a substrate is added into the culture parallel with the feeding step, and wherein the feeding step depends on the pH of the culture and its quantity is 0.5-1.0% related to the volume of the broth.  
   
   
       10 . The process of  claim 1 , wherein the fermentation step is carried out on a medium containing a carbon source selected from the group consisting of glucose, fructose and glycerine.  
   
   
       11 . The process of  claim 1 , wherein the fermentation step is carried out on a medium containing a nitrogen source selected from the group consisting of soybean meal, peptone, casein, yeast extract and meat extract.  
   
   
       12 . The process of  claim 2 , wherein the compound of formula (I) formed during the bioconversion is separated from the culture broth by adsorption from the filtrate of the broth and from the washing water of the mycelium on an anion exchange resin, eluting the compound of formula (I) from the resin, transforming the compound of formula (I) completely to its lactone form, isolating the lactone derivative, hydrolyzing the lactone derivative by sodium hydroxide, and desalting the compound of formula (I) on a non-ionic adsorption resin.  
   
   
       13 . The process of  claim 12 , wherein said anion exchange resin has quaternary ammonium active groups carrying polystyrene-divinylbenzene skeleton, is used for the separation of the compound of formula (I) from the filtrate of the broth.  
   
   
       14 . The process of  claim 4 , wherein the compound of formula (I) formed during the bioconversion is extracted in acid form from the broth, the broth having been acidified to the pH of 3.5-3.7, or from filtrate of the broth by a water-immiscible organic solvent.  
   
   
       15 . The process of  claim 14  wherein said water-immiscible organic solvent is ethyl acetate.  
   
   
       16 . The process of  claim 14  wherein said water immiscible organic solvent is isobutyl acetate.  
   
   
       17 . The process of  claim 5 , wherein said compound of formula (I) is extracted in sodium salt form from the organic solvent by aqueous sodium hydroxide solution, and purified on a non-ionic adsorption resin.  
   
   
       18 . The process of  claim 14 , wherein said compound of formula (I) is precipitated from the extract in crystalline form with a secondary amine containing alkyl-, cycloalkyl-, aralkyl- or aryl- substituents.  
   
   
       19 . The process of  claim 18 , wherein the crystalline secondary amine salt is suspended in a mixture of a 1-4 carbon atom-containing alkyl ester of acetic acid and water; an equivalent quantity of sodium hydroxide is added in aqueous solution to the suspension such that an organic phase and an aqueous phase are formed; the organic and aqueous phases are separated; the aqueous phase is washed with isobutyl acetate, then clarified with activated carbon; and the aqueous solution is lyophilized.  
   
   
       20 . The process of  claim 19 , wherein said alkyl ester is isobutyl ester.  
   
   
       21 - 33 . (canceled)