Method for producing l-beta-lysine

Abstract

Purified β-amino acids are of considerable interest in the preparation of pharmacologically active compounds. Although enantiomerically pure β-amino acids, such as L-β-lysine, can be produced by standard chemical synthesis, this traditional approach is time consuming, requires expensive starting materials, and results in a racemic mixture which must be purified further. However, DNA molecules encoding lysine 2,3-aminomutase can be used to prepare L-β-lysine by methods that avoid the pitfalls of chemical synthesis. In particular, L-β-lysine can be synthesized by cultures of host cells that express recombinant lysine 2,3-aminomutase. Alternatively, such recombinant host cells can provide a source for isolating quantities of lysine 2,3-aminomutase, which in turn, can be used to produce L-β-lysine in vitro.

1 . A method of producing L-β-lysine, comprising:
 (a) culturing a prokaryotic host cell comprising an expression vector that encodes lysine 2,3-aminomutase in the presence of L-lysine, wherein the vector that encodes lysine 2,3-aminomutase has a nucleic acid sequence of SEQ ID NO: 3 and the cultured host cell expresses lysine 2,3-aminomutase, and   (b) isolating L-β-lysine from the cultured host cells.   
     
     
         2 . The method of  claim 1  wherein the isolated L-β-lysine is enantiomerically pure. 
     
     
         3 . A method of producing L-β-lysine, comprising:
 (a) immobilizing lysine 2,3-aminomutase on a suitable support, wherein the lysine 2,3-aminomutase has the amino acid sequence of SEQ ID NO: 4;   (b) activating the lysine 2,3-aminomutase with cofactors required for lysine 2,3-aminomutase activity; and   (c) contacting L-lysine with the immobilized lysine 2,3-aminomutase to produce L-β-lysine.   
     
     
         4 . The method of  claim 3  wherein the L-lysine is contacted with the immobilized lysine 2,3-aminomutase for a sufficient amount of time to produce enantiomerically pure L-β-lysine. 
     
     
         5 . The method of  claim 2  further comprising separating the L-β-lysine from the L-lysine. 
     
     
         6 . The method of  claim 5  wherein the separation of the L-β-lysine from the L-lysine is achieved using high performance chromatography. 
     
     
         7 . The method of  claim 2  wherein the process is a continuous process. 
     
     
         8 . The method of  claim 3  wherein the cofactors required for lysine 2,3-aminomutase activity comprise:
 (i) at least one of ferrous sulfate or ferric ammonium sulfate;   (ii) pyridoxal phosphate;   (iii) at least one of dehydrolipoic acid, glutathione or dithiothreitol;   (iv) S-adenosylmethionine; and   (v) sodium dithionite.   
     
     
         9 . A method of producing L-β-lysine, comprising:
 (a) culturing a prokaryotic host cell comprising an expression vector that encodes lysine 2,3-aminomutase in the presence of L-lysine, wherein the cultured host cell expresses lysine 2,3-aminomutase, and   (b) isolating L-β-lysine from the cultured host cells, wherein the lysine 2,3-aminomutase has the amino acid sequence of SEQ ID NO: 4.   
     
     
         10 . A method of producing L-β-lysine, comprising:
 (a) incubating L-lysine in a solution containing purified lysine 2,3-aminomutase, wherein the lysine 2,3-aminomutase has the amino acid sequence of SEQ ID NO: 4, said solution containing all cofactors required for lysine 2,3-aminomutase activity; and   (b) isolating L-β-lysine from the incubation solution.   
     
     
         11 . The method of  claim 10 , wherein step (b) further comprises isolating L-β-lysine from L-lysine via chromatography.