Inline quantification of NADH und NADPH under process conditions (InCo)

Nicotine amide dinucleotide cofactors NADP+/NADPH and NAD+/NADH, respectively, are of paramount importance for the industrial utilisation of oxidoreductases. For the rational use a knowledge gap persists between predictions based on initial rate measurements and their application in processes. The aim of the project is the inline quantification of cofactors under conditions as close as possible to process conditions by application of fluorescence spectrometry allowing monitoring at µmol L^-1 concentrations which are common in their application (Fig. 1). The investigated enzymatic process is the enantioselective reduction of 2-octanone to (R)-2-octanol with LbADH with enzyme-coupled in-situ regeneration of the oxidized co-factor (NADP+) with GDH (Fig. 2). Systematic modelling will foster the understanding of the underlying mechanisms. A generally applicable method for kinetic characterisation of the reaction system will be derived. Ultimately, inline monitoring of continuous systems will be addressed. The findings expected as a result of the work are essential for the rational usage of cofactors in reductive in vitro biotransformations.

Figure 1: Different fluorescence properties of the oxidised (NADP+) and reduced (NADPH) forms of the cofactor

Figure 2: Reaction scheme for the enantioselective synthesis of (R)-2-octanol with LbADH and enzyme-coupled in-situ co-factor regeneration with GDH