Gluconobacter oxydans, an organism used for the industrial production of gluconic acid, contains two glucose dehydrogenases (GDHs) catalysing the direct oxidation of glucose to gluconic acid. In addition to a quinoprotein (PQQ-dependent), membrane-bound GDH (EC 1.1.99.17), an NADP-dependent, cytoplasmic GDH is present. From three types of experiments, evidence is presented that the quinoprotein GDH is the enzyme responsible for gluconic acid production by G. oxydans. In cell-free extracts, the activity of quinoprotein GDH was 30-fold higher than the activity of NADP-dependent GDH. A kinetic analysis of glucose-dependent oxygen uptake showed that a system with an affinity constant similar to the Km of quinoprotein GDH is involved in the process of gluconic acid production. The conclusion that gluconic acid production is essentially an extracytoplasmic process catalysed by quinoprotein GDH was confirmed in uptake experiments, which demonstrated that inhibition of glucose transport does not result in inhibition of gluconic acid production.@en