Guidelines for the Application of NAD(P)H Regenerating Glucose Dehydrogenase in Synthetic Processes

Abstract

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.

Mutagenesis‐Independent Stabilization of Class B Flavin Monooxygenases in Operation

“…Here,N ADP + + and an enzymatic cofactorr ecycling systemw as set in place.T he sacrificials ubstrate glucose was supplied in sufficient excess (! [36] Increasing the stabilizing power of the formulation (addition of FAD, ROS enzymes,a nd more NADPH) led to as trong improvement of t 1/2 under turnover conditions.W eo bserved am uch higher stabilizing effect of FADa nd NADPH without CATand SOD in this assay form than in the incubatione xperiments (322 min vs. 99 min, Figure 5b,C onditionsbvs. [36] Increasing the stabilizing power of the formulation (addition of FAD, ROS enzymes,a nd more NADPH) led to as trong improvement of t 1/2 under turnover conditions.W eo bserved am uch higher stabilizing effect of FADa nd NADPH without CATand SOD in this assay form than in the incubatione xperiments (322 min vs. 99 min, Figure 5b,C onditionsbvs.…”

Engineering an enzymatic regeneration system for NAD(P)H oxidation

“…The rate constants with the laccase at pH 5.0, were high enough to use the selected laccase together with GDH. The catalytic performance of GDH at pH 5.0 compared to pH 8.0 [24] is still good enough to allow its application. One potential problem arising from employing GDH at low pH is the low hydrolysis rate of the reaction product gluconolactone, which results in product inhibition.…”

“…Laccase activity is modeled by a ping-pong bi-bi reaction mechanism [23] although this is only an approximation of the more complicated reaction mechanism for which no kinetic model is published. GDH activity was modeled by sequential ordered bi-bi mechanism kinetics with the coenzyme binding first [24]. The product of the laccase reaction, the oxidized redox mediator, reacts with one of the products of the GDH reaction, the reduced nicotinamide coenzyme, in a second-order reaction [5] (this work).…”

3.9 Scale-Up and Development of Enzyme-Based Processes for Large-Scale Synthesis Applications

“…Such systems may also be limited by the presence of inhibitory cosubstrate(s) and coproduct(s), the cost of the cosubstrate(s), as well as the cost of the additional enzymes. [33] Additionally, the cofactors themselves can be sensitive to the conditions. For example, all forms of the nicotinamide cofactors are sensitive to pH, bringing obvious limitations to the process.…”