Characterization and Application of an Aspartate Dehydrogenase from Achromobacter denitrificans.

A novel gene encoding aspartate dehydrogenase (ASPDH) has been discovered in Achromobacter denitrificans. The product of this gene has a strict dependence on NADH and demonstrated significant reductive activity towards not only oxaloacetate (OAA) but also 2-ketobutyric acid. Further enzymatic characterization revealed the kinetic parameters of ASPDH for OAA and 2-ketobutyric acid were as follows: K m values of 4.25 mM and 0.89 mM, V max values of 10.67 U mg -1 and 2.10 U mg -1 , and K cat values of 3.70 s -1 and 0.72 s -1 , respectively. The enzyme also showed a dependency on metal ions, with EDTA and Cu 2+ exerting strong inhibitory effects, while Ca 2+ and Fe 2+ exhibited pronounced enhancing effects. By utilizing a whole-cell biocatalyst system comprising glucose dehydrogenase (GDH) and ASPDH as a coupled system to replenish cofactors by oxidizing glucose, enabling the effective conversion of 2-ketobutyric acid to L-2-aminobutyric acid (L-2-ABA) with 97.2% yield.