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 Cu²⁺ exerting strong inhibitory effects, while Ca²⁺ and Fe²⁺ 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.