Next, we further investigated the NR-triggered conversion of 1a to 6a'. First, we investigated whether the NR and glucose dehydrogenase (GDH) concentrations in the reaction with 1a could be lowered without prolonging the reaction time required for a good conversion (Figure ). Reducing the Bacillus megaterium GDH (BmGDH) concentration from 8 µM to 0.8 µM had no ebect on the course of the reaction, which led us to reduce the BmGDH concentration to 1.6 µM for all further experiments. In contrast, reducing the concentration of NfsA or BaNTR1 resulted in a significant change in the product formation after one hour. Considering that some substrates might lead to lower activity when screening the substrate range, the standard concentration of NR protein was kept at 20 µM. In addition, we found that the 50 mM phosphate buber at pH 7.0 used in all previous experiments was not subicient to prevent the buber from going into the acidic range during the reaction. This was caused by the gluconic acid formed during the cofactor regeneration cycle. By increasing the buber strength to 200 mM and raising the pH to 8.0, it was possible to achieve higher or even complete conversion to 6a' with BaNTR1 and NfsA, respectively (Figure ).