Substrate selectivity of Gluconobacter oxydans for production of 2,5-diketo-D-gluconic acid and synthesis of 2-keto-L-gulonic acid in a multienzyme system

Substrate selectivity of Gluconobacter oxydans (ATCC 9937) for 2,5-diketo-D -gluconic acid (2,5-DKG) production was investigated with glucose, gluconic acid, and gluconolactone in different concentrations using a resting-cell system. The results show that gluconic acid was utilized favorably by G. ox ydans as substrate to produce 2,5-DKG. The strain was coupled with glucose dehydrogenase (GDH) and 2,5-DKG reductase for synthesis of 2-keto-L-gulonic acid (2-KLG), a direct precursor Of L-ascorbic acid, from glucose. NADP an d NADPH were regenerated between GDH and 2,5-DKG reductase. The mole yield of 2-KLG of this multienzyme system was 16.8%. There are three advantages f or using the resting cells of G. oxydans to connect GDH with 2,5-DKG reduct ase for production of 2-KLG: gluconate produced by GDH may immediately be t ransformed into 2,5-DKG so that a series of problems generally caused by th e accumulation of gluconate would be avoided; 2,5-DKG is supplied directly and continuously for 2,5-DKG reductase, so it is unnecessary to take specia l measures to deal with this unstable substrate as it was in Sonoyama's tan dem fermentation process; and NADP(H) was regenerated within the system wit hout any other components or systems.