Glucose Dehydrogenase for the Regeneration of NADPH and NADH

Glucose dehydrogenases (GDHs) occur in several organisms such as Bacillus megaterium and Bacillus subtilis. They accept both NAD+and NADP+as cofactor and can be used for the regeneration of NADH and NADPH. In order to demonstrate their applicability we coupled an NADP+-dependent, (R)-specific alcohol dehydrogenase (ADH) from Lactobacillus kefir with the glucose dehydrogenase from B. subtilis. The ADH reduces prochiral ketones stereoselectively to chiral alcohols. The reduction requires NADPH, which was regenerated by the glucose dehydrogenase. Glucose dehydrogenase from B. subtilis (EC 1.1.1.47) is a tetramer with a molecular weight of 126,000. The enzyme shows a pH optimum at 8.0 and a broad temperature optimum at 45-50°C. We investigated the conversion of acetophenone in a cell-free system with purified ADH and GDH. Furthermore, we constructed two plasmids containing the genes encoding ADH and GDH by inserting them one after the other. These two plasmids differ from each other in the order of the genes. Because of the low solubility of the compounds, we examined the reaction in a water/organic solvent two-phase system.