On the Use of the Adsorption Energy Distribution for the Analysis of Competing Substrate Kinetics

Abstract

The use of renewable raw materials poses new challenges for the chemical and biotechnological industry. For the analysis of the reaction kinetics, elaborate procedures are necessary as the materials are more complex. Thus, a question arises whether interdisciplinary approaches may help to meet these challenges. In this study, the estimation of the adsorption energy distribution (AED) is used to characterize enzyme kinetics and, in particular, competing substrate kinetics for the example of xylose isomerase. Therefore, the kinetic parameters of two literature models are determined based on nonlinear regression and compared with parameter estimates derived from the estimation of the AED. It was shown that based on the AED, the number of competing substrates can be identified automatically, while this information must be provided a priori for the correct identification of the parameters of the literature models via direct nonlinear regression. Thus, the AED provides a promising tool for the characterization of competing substrate kinetics.

Using Adsorption Energy Distribution for Parameter Estimation of Competitive Cofactor Coupled Enzyme Reaction

“…Equation (1). Refer to the original work for the uni-uni reaction [25] for a more thorough description of the fundamentals.…”

“…Details are provided in the supporting information. For further analysis of the results, the standard error of the estimates was calculated manually according to our previous work [25].…”

“…Remarkably, the NLR predicts the same K m values for 2-butanol and propanol and the corresponding co-factor. However, the NLR estimates depend considerably on the initial values, which is a well-known problem when utilizing local optimization algorithms, such as the applied Levenberg-Marquardt algorithm [25,41]. The NLR estimates for alternative initial values are listed in Table S3.…”

“…Further research may use a model-based design of experiments to reduce the standard error of estimated parameters and to support model discrimination [46]. Further research may address an independent validation of the experimental results by discrimination of different alcohols in the analysis, as was demonstrated for a simple uni-uni reaction via HPLC before [25]. For the given experimental set-up, an offline analytic is not reasonable, as the measuring sample cannot be taken by a closed spectrometer, and the reaction takes place within seconds.…”

“…The approach is based on the estimation of the adsorption energy distribution (AED), which allows for the identification of the number of individual substrates and an estimation of the kinetic parameters of different substrates in a mixture for an enzymatic reaction using the non-selective measurement of co-factor consumption. The results of the proposed AED-based method, which was introduced and validated for single-substrate enzymatic reactions in our preceding work [25], are compared with parameter estimates obtained from classical non-linear regression based on concentration measurements. As was previously demonstrated, the AED-based method requires only a small number of composition measurements while avoiding the need for costly separation of individual products, such as through high-performance liquid chromatographic (HPLC) methods.…”