(175ar) An Innovative Method for the Characterization of Multi-Substrate Enzyme Reactions

The analysis of complex (bio)chemical processes is a major challenge in process engineering. This applies in particular for “green” and “sustainable” processes, because they deal with complex substrates, reaction sequences or new materials. Interdisciplinary approaches may help to develop new and innovative solutions.

Here we present a study with an innovative and interdisciplinary method for the characterization of multi-substrate enzyme reactions. The scope of this research is to apply the method of adsorption energy distribution (AED) to be used for the characterization of enzymatic processes. The AED was originally developed to analyze heterogeneous surfaces. However, this method can also be used to determine different binding sites of an adsorptive on homogeneous surfaces and can be flexibly adapted with different mathematical models for the description of the adsorption isotherm.

For the example of an alcohol dehydrogenase, we present the analysis of pure substrate ethanol and pure substrate butanol as well as the analysis of a mixture of both alcohols. In all cases the absorption of co-factor NADH was used for the determination of the reaction kinetics. The analysis was carried out with a a non-linear regression for the Michaelis-Menten equation as well as with AED-Analysis. For pure substrates there were satisfactory similarities between non-linear regression and AED-Analysis in respect to K_M and v_max. Using standard methods, the mixture could not be analyzed by the NADH absorption, because NADH does not distinguish between ethanol and butanol. However, with our new approach this is possible. The results show two individual reactions kinetics: two K_M-values as well as two v_max-values are calculated by analyzing the NADH absorption. Thus it is possible to determine the apparent parameters for a multi-substrate enzyme reaction with the determination of the same co-factor.