Microbiological method for producing amides

Abstract

The present invention provides for microorganisms capable of tolerating acetonitrile concentrations of at least 3 M, enzyme extracts obtainable from these microorganisms, a nitrile hydratase obtainable from these microorganisms, a method for preparing amides using these microorganisms, the enzyme extracts or the nitrile hydratase obtainable therefrom, and a method for removing acetonitrile from solutions using these microorganisms, the enzyme extracts or the nitrile hydratase obtainable therefrom.

What is claimed is:  
     
         1 . A microorganism capable of tolerating an acetonitrile concentration of at least 3 M.  
     
     
         2 . The microorganism of  claim 1 , wherein said microorganism is a member of the genus Rhodococcus.  
     
     
         3 . The microorganism of  claim 2 , wherein said microorganism is a member of the strain Rhodococcus sp. FZ4, deposited under the DSM number 13597.  
     
     
         4 . An enzyme extract obtainable from the microorganisms of any one of  claims 1  to  3 .  
     
     
         5 . A nitrile hydratase obtainable from the microorganisms of any one of  claims 1  to  3 .  
     
     
         6 . The nitrile hydratase of  claim 5 , wherein said nitrile hydratase exhibits 
 a) a K M  value of 2.84±1.00 mM for the substrate acetonitrile and of 80.5±15.0 mM for the substrate 3-cyanopyridine; and    b) a pH optimum of pH 6.5±1.0.    
     
     
         7 . A nitrile hydratase, wherein said nitrile hydratase exhibits 
 a) a KM value of 2.84±1.00 mM for the substrate acetonitrile and of 80.5±15.0 mM for the substrate 3-cyanopyridine;    b) a pH optimum of pH 6.5±1.0.    
     
     
         8 . A method for preparing amides of the general formula  
       R 1 —CONH 2    III  
       wherein R 1  is selected from the group consisting of a C 1-6 -alkyl radical, a C 2-6 -alkenyl group or a group of the general formula  
       
         
           
           
               
               
           
         
         wherein X is a nitrogen atom or —CH═,  
         wherein R 2  and R 3  are selected from the group consisting of a hydrogen atom, a halogen atom, a C 1-6 -alkyl group or a C 2-6 -alkenyl group,  
         wherein said method a nitrile of the general formula  
         R 1 —CN   II,  
         in which R 1  is selected from the group consisting of a C 1-6 -alkyl radical, a C 2-6 -alkenyl group or a group of the general formula  
         
           
             
             
                 
                 
             
           
         
         is converted by means of the microorganism of any one of  claim 1  to  3 .  
       
     
     
         9 . The method of  claim 8 , wherein the nitrile is 3-cyanopyridine or acetonitrile.  
     
     
         10 . The method of  claim 8 , wherein the reaction is carried out at a temperature of from 5 to 50° C. and at a pH of from about 5 to 10.  
     
     
         11 . The method of  claim 9 , wherein the reaction is carried out at a temperature of from 5 to 50° C. and at a pH of from about 5 to 10.  
     
     
         12 . A method for preparing amides of the general formula  
       R 1 —CONH 2    III  wherein R 1  is selected from the group consisting of a C 1-6 -alkyl radical, a C 2-6 -alkenyl group or a group of the general formula                          wherein X is a nitrogen atom or —CH═,    wherein R 2  and R 3  are selected from the group consisting of a hydrogen atom, a halogen atom, a C 1-6 -alkyl group or a C 2-6 -alkenyl group,    wherein said method a nitrile of the general formula    R 1 —CN   II,    in which R 1  is selected from the group consisting of a C 1-6 -alkyl radical, a C 2-6 -alkenyl group or a group of the general formula                          is converted by means of an enzyme extract obtainable from the microorganisms of any one of  claims 1  to  3 .    
     
     
         13 . The method of  claim 12 , wherein the nitrile is 3-cyanopyridine or acetonitrile.  
     
     
         14 . The method of  claim 12 , wherein the reaction is carried out at a temperature of from 5 to 50° C. and at a pH of from about 5 to 10.  
     
     
         15 . The method of  claim 13 , wherein the reaction is carried out at a temperature of from 5 to 50° C. and at a pH of from about 5 to 10.  
     
     
         16 . A method for preparing amides of the general formula  
       R 1 —CONH 2    III  
       wherein R 1  is selected from the group consisting of a C 1-6 -alkyl radical, a C 2-6 -alkenyl group or a group of the general formula  
       
         
           
           
               
               
           
         
       
       wherein X is a nitrogen atom or —CH═, 
 wherein R 2  and R 3  are selected from the group consisting of a hydrogen atom, a halogen atom, a C 1-6 -alkyl group or a C 2-6 -alkenyl group,  
 wherein said method a nitrile of the general formula  
 R 1 —CN   II,  
 in which R 1  is selected from the group consisting of a C 1-6 -alkyl radical, a C 2-6 -alkenyl group or a group of the general formula  
                     
 is converted by means of the nitrile hydratase obtainable from the microorganisms of any one of  claims 1  to  3 .  
 
     
     
         17 . The method of  claim 16 , wherein the nitrile is 3-cyanopyridine or acetonitrile.  
     
     
         18 . The method of  claim 16 , wherein the reaction is carried out at a temperature of from 5 to 50° C. and at a pH of from about 5 to 10.  
     
     
         19 . The method of  claim 17 , wherein the reaction is carried out at a temperature of from 5 to 50° C. and at a pH of from about 5 to 10.  
     
     
         20 . A method for preparing amides of the general formula  
       R 1 —CONH 2    III  
       wherein R 1  is selected from the group consisting of a C 1-6 -alkyl radical, a C 2-6 -alkenyl group or a group of the general formula  
       
         
           
           
               
               
           
         
       
       wherein X is a nitrogen atom or —CH═, 
 wherein R 2  and R 3  are selected from the group consisting of a hydrogen atom, a halogen atom, a C 1-6 -alkyl group or a C 2-6 -alkenyl group,  
 wherein said method a nitrile of the general formula  
 R 1 —CN   II,  
 in which R 1  is selected from the group consisting of a C 1-6 -alkyl radical, a C 2-6 -alkenyl group or a group of the general formula  
                     
 is converted by means of a nitrile hydratase, said nitrile hydratase exhibiting  
 a) a K M  value of 2.84±1.00 mM for the substrate acetonitrile and of 80.5±15.0 mM for the substrate 3-cyanopyridine; and  
 b) a pH optimum of pH 6.5±1.0.  
 
     
     
         21 . The method of  claim 20 , wherein the nitrile is 3-cyanopyridine or acetonitrile.  
     
     
         22 . The method of  claim 20 , wherein the reaction is carried out at a temperature of from 5 to 50° C. and at a pH of from about 5 to 10.  
     
     
         23 . The method of  claim 21 , wherein the reaction is carried out at a temperature of from 5 to 50° C. and at a pH of from about 5 to 10.  
     
     
         24 . A method for removing acetonitrile waste comprising the microorganisms of any one of  claims 1  to  3 .  
     
     
         25 . A method for removing acetonitrile waste comprising the an enzyme extract obtainable from the microorganisms of any one of  claims 1  to  3 .  
     
     
         26 . A method for removing acetonitrile waste comprising the nitrile hydratase obtainable from the microorganisms of any one of  claims 1  to  3 .