Glucose dehydrogenase (GDH) is frequently used for the reduction of NAD + and NADP + in bench-and industrial-scale syntheses because the coenzyme regenerating system GDH is easy to apply, robust and relatively inexpensive. To optimize the application of this long known coenzyme regeneration system we investigated the commonly applied Bacillus GDH and characterized this enzyme by its kinetic features in the presence of substrates and products at pH 6.4 and 8.0. Three substrates/products were found to inhibit GDH considerably: (i) the reaction product glucono-1,5-lactone, (ii) the reduced coenzyme NAD(P)H and (iii) the oxidized coenzyme NAD(P)+ . The inhibition of GDH under several process conditions was modeled using the determined kinetic constants. It was found that the GDH regeneration system is strongly inhibited by the usually applied conditions. This study provides the rate equation of the GDH reaction and simulations of this coenzyme regenerating system leading to an improved prediction and, thus, to a faster scale-up and increased efficiency of NAD(P)H-dependent synthetic processes.

2,5-diketo-D-gluconic acid reductase (2,5-DKG reductase) catalyses the reduction of 2,5-diketo-D-gluconic acid (2,5-DKG) to 2-keto-L-gulonic acid (2-KLG), a direct precursor (lactone) of L-ascorbic acid (vitamin C). This reaction is an essential step in the biocatalytic production of the food supplement vitamin C from D-glucose or D-gluconic acid. As 2,5-DKG reductase is usually produced recombinantly, it is of interest to establish an efficient process for 2,5-DKG reductase production that also satisfies food safety requirements. In the present study, three recently described food grade variants of the Lactobacillales based expression systems pSIP (Lactobacillus plantarum) and NICE (Lactococcus lactis) were evaluated with regard to their effictiveness to produce 2,5-DKG reductase from Corynebacterium glutamicum. Our results indicate that both systems are suitable for 2,5-DKG reductase expression. Maximum production yields were obtained with Lb. plantarum/pSIP609 by pH control at 6.5. With 262 U per litre of broth, this represents the highest heterologous expression level so far reported for 2,5-DKG reductase from C. glutamicum. Accordingly, Lb. plantarum/pSIP609 might be an interesting alternative to Escherichia coli expression systems for industrial 2,5-DKG reductase production.

No abstract

No abstract