Production and Purification of 5-Keto-D-Fructose

To produce 5-KF from D-fructose, a cryopreserved stock of G. oxydans fdh was streaked on YMF-plates (section “Shake Flask Cultivation of Microbial Strains”) containing 1.5% [w/v] Agar-Agar, Kobe I, and the antibiotics cefoxitin and kanamycin at final concentrations of 50 μg mL^–1. To evaluate the impact of potential cellular contamination during 5-KF production on subsequent toxicological studies, a reference fructose solution was prepared using G. oxydans 621H ΔhsdR, which cannot oxidize D-fructose in the form of dormant cells. Thus, apart from potential contaminations with cellular components, a D-fructose solution emerges unchanged from whole-cell catalysis with resting cells of G. oxydans 621H ΔhsdR. After 48 h of incubation at 30^°C, single colonies of each strain were used to inoculate 50 mL of YMF-precultures supplemented with the required antibiotics (see section “Shake Flask Cultivation of Microbial Strains”). Precultures were maintained in shake flasks at 30^°C and 200 rpm for 48 h for the subsequent inoculation of the main cultures. Therefore, 250 mL of YMF medium were inoculated with 12.5 mL of preculture and incubated for 24 h at 30^°C and 200 rpm. The cultivation was carried out in 2-L shake flasks to ensure sufficient oxygen supply. After cultures were harvested by centrifugation (8,000 × g, 10^°C, 15 min), cell pellets were resuspended in 10 mL of 50 mM D-fructose solution and again centrifuged, applying the same conditions. This washing procedure was repeated two more times before the cells were finally resuspended in 5 ml of 50 mM D-fructose solution. Bioconversion of D-fructose to 5-KF was carried out at a 250-mL scale in 2-L shake flasks, using a 200 mM D-fructose solution adjusted to pH 6 by adding 5 mM of MES-buffer (pH 6). The washed cells of G. oxydans 621H ΔhsdR and G. oxydans fdh were added to separate flasks and incubated for 18 h at 30^°C and 200 rpm. After cell removal by centrifugation (10,000 × g, 20^°C, 20 min), the supernatants were treated with activated charcoal (Cabot Norit GAC 1240 Plus; Cabot Corporation, US, Boston). Therefore, 26 mg of activated charcoal were added per mL supernatant and incubated under stirring for 1 h at 22^°C. Subsequently, the suspension was subjected to a final centrifugation step (10,000 × g, 20^°C, 10 min). The generated supernatants were first filtered through CHROMAFIL^® RC-45/25 syringe filters (0.45 μm pore size) and sterilized by subsequent filtration using sterile PVDF syringe filters (0.22 μm pore size). Filtrates were stored at 8^°C in sterile falcon tubes.