l-2-hydroxyisocaproate dehydrogenase?A new enzyme from Lactobacillus confusus for the stereospecific reduction of 2-ketocarboxylic acids

A new dehydrogenase from Lactobacillus confusus has been purified. The following reactions are catalyzed by this enzyme: $$\vec F_\alpha$$ Various 2-ketocarboxylic acids are stereospecifically reduced to the corresponding l-2-hydroxycarboxylic acids. In the reverse reaction the NAD dependent dehydrogenation of l-2-hydroxycarboxylic acids is observed. l-2-hydroxyisocaproate seems to be the best substrate for this enzyme which we therefore call l-2-hydroxyisocaproate dehydrogenase (l-HicDH). The enzyme requires NAD(H) as a cofactor, which cannot be replaced by NADP(H). The large scale purification of the enzyme is described starting with 24 kg wet cells, it involved homogenization using a continuously operating high speed bead mill, liquid-liquid extraction with aqueous two-phase systems and diafiltration followed by ion-exchange chromatography on DEAE-cellulose. At this stage the enzyme was purified 177-fold resulting in a specific activity of 30 U/mg. This technical catalyst can be used for the continuous production of l-2-hydroxycarboxylic acids in an enzyme membrane reactor. Further purification up to 450–480 U/mg can be carried out by interfacial salting out chromatography or affinity chromatography, respectively. Properties of the purified enzyme were investigated in detail especially the parameters which are important for an industrial application.