Enzymatic synthesis of l-?-chloroalanine using amino acid dehydrogenase

Abstract

l-β-Chloroalanine is a useful intermediate for the synthesis of several l-amino acids. Conditions for synthesizing optically pure l-β-chloroalanine from 3-chloropyruvate using alanine dehydrogenase (AlaDH), leucine dehydrogenase and phenylalanine dehydrogenase with a regeneration of NADH by formate dehydrogenase (FDH) were investigated. The enzymatic reaction was carried out at neutral pH because of a chemical instability of 3-chloropyruvate on the alkaline side. Commercially available AlaDH from Bacillus stearothermophilus IFO 12550 showed the highest activity for the production of l-β-chloroalanine at pH 7.5. The K m and V max values for 3-chloropyruvate of AlaDH were calculated to be 300 units/mg and 62.5 mm, respectively. Although 3-chloropyruvate had no inhibitory effect on AlaDH, it acted as a non-competitive inhibitor with FDH. 3-Chloropyruvate was added into the reaction mixture in a stepwise manner to avoid the inhibition. l-β-Chloroalanine was produced with high chemical (>90%) and optical yields (100% enantiometric excess) and at a high concentration (43 g/l).