(r)- selective amination

Abstract

The present invention relates to a method for the enzymatic synthesis of enantiomerically enriched (R)-amines of general formula [1][c], from the corresponding ketones of general formula [1][a], by using novel transaminases. These novel transaminases are selected from two different groups: either from a group of some 20 proteins with sequences as specified herein, or from a group of proteins having transaminase activity and isolated from a microorganism selected from the group of organisms consisting of Rahnella aquatilis, Ochrobactrum anthropi, Ochrobactrum tritici, Sinorhizobium morelense, Curtobacterium pusilllum, Paecilomyces lilacinus, Microbacterium ginsengisoli, Microbacterium trichothecenolyticum, Pseudomonas citronellolis, Yersinia kristensenii, Achromobacter spanius, Achromobacter insolitus, Mycobacterium fortuitum, Mycobacterium frederiksbergense, Mycobacterium sacrum, Mycobacterium fluoranthenivorans, Burkholderia sp., Burkholderia tropica, Cosmospora episphaeria , and Fusarium oxysporum .

1 . A method for the enzymatic synthesis of an enantiomerically enriched (R)-amine of general formula [1][c] 
       
         
           
           
               
               
           
         
       
       from the corresponding ketone of the general formula 
       
         
           
           
               
               
           
         
       
       and a suitable amino donor, wherein R 1  and R 2  are different and are independently linear or branched aliphatic, aromatic, hetero-aromatic or form a cyclic structure, by using a transaminase selected from the group consisting of
 a. a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 1; 
 b. a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 3; 
 c. a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 5; 
 d. a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 7; 
 e. a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 9; 
 f. a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 11; 
 g. a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 13; 
 h. a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 15; 
 i. a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 17; 
 j. a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 19; 
 k. a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 21; 
 l. a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 23; 
 m. a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 25; 
 n. a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 30; 
 o. a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 33; 
 p. a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 35; 
 q. a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 38; 
 r. a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 40; 
 s. a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 43; 
 t. a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 46 and 
 u. a protein having transaminase activity and obtainable from an organism selected from the group of organisms consisting of  Rahnella aquatilis, Ochrobactrum anthropi, Ochrobactrum tritici, Sinorhizobium morelense, Curtobacterium pusilllum, Paecilomyces lilacinus, Microbacterium ginsengisoli, Microbacterium trichothecenolyticum, Pseudomonas citronellolis, Yersinia kristensenii, Achromobacter spanius, Achromobacter insolitus, Mycobacterium fortuitum, Mycobacterium frederiksbergense, Mycobacterium sacrum, Mycobacterium fluoranthenivorans, Burkholderia  sp.,  Burkholderia tropica, Cosmospora episphaeria , and  Fusarium oxysporum.    
 
     
     
         2 . A method according to  claim 1 , wherein R 1  and R 2  are different and R 1  and R 2  independently contain 1 to 30 carbon atoms and R 1  and R 2  are independently substituted or unsubstituted aliphatic; substituted or unsubstituted branched aliphatic; substituted or unsubstituted cyclic aliphatic; substituted or unsubstituted heterocyclic aliphatic, containing at least one oxygen, sulfur or nitrogen atom; substituted or unsubstituted aromatic; substituted or unsubstituted hetero-aromatic containing at least one sulfur or nitrogen atom; or together form a substituted or unsubstituted cyclic structure or heterocyclic structure, containing at least one oxygen, sulfur or nitrogen atom; wherein the substituents are selected from but not limited to the group consisting of a halogen atom, an alkyl group having 1 to 6 carbon atoms, hydroxyl group, methoxy group, monofluoromethyl, difluoromethyl and trifluoromethyl group. 
     
     
         3 . A process according to  claim 1 , characterized in that the final concentration of the enantiomerically enriched (R)-amine product lies between 1 and 50 weight % of the reaction mixture. 
     
     
         4 . A process according to  claim 3 , wherein the transaminase is a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 1. 
     
     
         5 . A process according to  claim 4 , wherein the amino donor is α-methylbenzylamine. 
     
     
         6 . A process according to  claim 4 , wherein the amino donor is sec-butylamine. 
     
     
         7 . A process according to  claim 1 , characterized in that the final concentration of the enantiomerically enriched (R)-amine product lies between 5 and 35 weight % of the reaction mixture. 
     
     
         8 . A process according to  claim 1 , wherein the reaction mixture comprises an aqueous phase and second organic phase. 
     
     
         9 . A process according to  claim 1 , wherein the reaction mixture comprises an aqueous phase and second organic phase and the volumetric ratio of water:organic phase is between 100 and 0.01. 
     
     
         10 . A process according to  claim 1 , wherein the reaction mixture comprises an aqueous phase and second organic phase and the volumetric ratio of water:organic phase is between 20 and 0.1. 
     
     
         11 . A process according to  claim 1 , wherein the reaction mixture comprises an aqueous phase and second organic phase and the volumetric ratio of water:organic phase is between 20 and 1. 
     
     
         12 . A microorganism comprising an enzyme having (R)-transaminase activity from the group of organisms consisting of  Rahnella aquatilis  (deposited as DSM 23797),  Ochrobactrum anthropi  (deposited as DSM 23793),  Ochrobactrum tritici  (deposited as DSM 23786),  Sinorhizobium morelense  (deposited as DSM 23794),  Curtobacterium pusilllum  (deposited as DSM 23787),  Paecilomyces lilacinus  (deposited as DSM 23771),  Microbacterium ginsengisoli, Microbacterium trichothecenolyticum  (deposited as DSM 23788),  Pseudomonas citronellolis  (deposited as DSM 23795),  Yersinia kristensenii  (deposited as DSM 23792),  Achromobacter spanius  (deposited as DSM 23791),  Achromobacter insolitus  (deposited as DSM 23790),  Mycobacterium fortuitum  (deposited as DSM 23789),  Mycobacterium frederiksbergense  (deposited as DSM 23798),  Mycobacterium sacrum  (deposited as DSM 23785),  Mycobacterium fluoranthenivorans  (deposited as DSM 23796),  Burkholderia  sp.,  Burkholderia tropica  (deposited as DSM 23799),  Cosmospora episphaeria  (deposited as DSM 23772), and  Fusarium oxysporum  (deposited as DSM 23770). 
     
     
         13 . A microorganism according to  claim 12 , wherein the enzyme having (R)-transaminase activity is selected from the group consisting of Cpu-TA1 [SEQ ID No. 30], Cpu-TA2 [SEQ ID No. 33], Cpu-TA3 [SEQ ID No. 35], Raq-TA2 [SEQ ID No. 38], Raq-TA3 [SEQ ID No. 40], Asp-TA1 [SEQ ID No. 43] and Mgi-TA1 [SEQ ID No. 46] or a protein having at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% identity to any of these amino acid sequences. 
     
     
         14 . A polypeptide having (R)-transaminase activity and obtainable from an organism selected from the group of organisms consisting of  Rahnella aquatilis  (deposited as DSM 23797),  Ochrobactrum anthropi  (deposited as DSM 23793),  Ochrobactrum tritici  (deposited as DSM 23786),  Sinorhizobium morelense  (deposited as DSM 23794),  Curtobacterium pusilllum  (deposited as DSM 23787),  Paecilomyces lilacinus  (deposited as DSM 23771),  Microbacterium ginsengisoli, Microbacterium trichothecenolyticum  (deposited as DSM 23788),  Pseudomonas citronellolis  (deposited as DSM 23795),  Yersinia kristensenii  (deposited as DSM 23792),  Achromobacter spanius  (deposited as DSM 23791),  Achromobacter insolitus  (deposited as DSM 23790),  Mycobacterium fortuitum  (deposited as DSM 23789),  Mycobacterium frederiksbergense  (deposited as DSM 23798),  Mycobacterium sacrum  (deposited as DSM 23785),  Mycobacterium fluoranthenivorans  (deposited as DSM 23796),  Burkholderia  sp.,  Burkholderia tropica  (deposited as DSM 23799),  Cosmospora episphaeria  (deposited as DSM 23772), and  Fusarium oxysporum  (deposited as DSM 23770). 
     
     
         15 . A polypeptide having (R)-transaminase activity and at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to a polypeptide having (R)-transaminase activity and obtainable from an organism selected from the group of organisms consisting of  Rahnella aquatilis  (deposited as DSM 23797),  Ochrobactrum anthropi  (deposited as DSM 23793),  Ochrobactrum tritici  (deposited as DSM 23786),  Sinorhizobium morelense  (deposited as DSM 23794),  Curtobacterium pusilllum  (deposited as DSM 23787),  Paecilomyces lilacinus  (deposited as DSM 23771),  Microbacterium ginsengisoli, Microbacterium trichothecenolyticum  (deposited as DSM 23788),  Pseudomonas citronellolis  (deposited as DSM 23795),  Yersinia kristensenii  (deposited as DSM 23792),  Achromobacter spanius  (deposited as DSM 23791),  Achromobacter insolitus  (deposited as DSM 23790),  Mycobacterium fortuitum  (deposited as DSM 23789),  Mycobacterium frederiksbergense  (deposited as DSM 23798),  Mycobacterium sacrum  (deposited as DSM 23785),  Mycobacterium fluoranthenivorans  (deposited as DSM 23796),  Burkholderia  sp.,  Burkholderia tropica  (deposited as DSM 23799),  Cosmospora episphaeria  (deposited as DSM 23772), and  Fusarium oxysporum  (deposited as DSM 23770). 
     
     
         16 . A polypeptide according to  claim 14  having (R)-transaminase activity selected from the group consisting of Cpu-TA1 [SEQ ID No. 30], Cpu-TA2 [SEQ ID No. 33], Cpu-TA3 [SEQ ID No. 35], Raq-TA2 [SEQ ID No. 38], Raq-TA3 [SEQ ID No. 40], Asp-TA1 [SEQ ID No. 43] and Mgi-TA1 [SEQ ID No. 46] or a protein having at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% identity to any of these amino acid sequences. 
     
     
         17 . A nucleic acid encoding for a polypeptide according to  claim 14 .