Influence of the experimental setup on the determination of enzyme kinetic parameters

Abstract

For the design of bioconversion processes parallel experimentation in microtiter plates is commonly applied to reduce the experimental load, although data accuracy and reproducibility are often reduced. In an effort to quantify the impact of different microscale experimental systems on the estimation of enzyme kinetic parameters from progress curves, we comprehensively evaluated the enzymatic reduction of acetophenone in both open and closed polystyrene and quartz microtiter plates as well as quartz cuvettes. Differences in conversion of up to 50% over time were observed increasing from polystyrene MTPs to quartz MTPs to quartz cuvettes. Initial reaction velocities increased systematically from polystyrene to quartz MTPs and cuvettes. The experimental errors decreased in the same order showing highest experimental error of about 20% in polystyrene. We further evaluated reasons causing the deviations within one system as well as between the systems. The choice of reaction vessel material, temperature effects and substrate cross contaminations in MTPs were shown to be of importance in the experimental results. Although the experimental data differed between the reaction vessels, no distinct trends in estimated kinetic parameters were found. While the microkinetic parameters vary up to an order of magnitude between different systems, the corresponding macrokinetic parameters lie in the same range for all systems varying by 29-118%. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:87-95, 2017.

Multi‐enzyme cascade reaction in a miniplant two‐phase‐system: Model validation and mathematical optimization

“…In Figure 4 Throughout all six experiments we simulated for model validation, the mean deviation reached values of 26.0% and 22.6% for COH and CCI respectively, and 11.66% for the overall molar balance, which can be traced back to the deviations of the enzymes' kinetic parameters. 26,[33][34][35] In literature, mathematical models for such complex multienzyme processes across phase boundaries in miniplant-scale and continuous operation cannot be found to the best of our knowledge.…”

Multi-Enzyme Cascade Reaction in a Miniplant Two-Phase-System: Model Validation and Mathematical Optimization

“…The experiment shown in Figure 4 In Figure 4 ters. 26,[33][34][35] In literature, mathematical models for such complex multienzyme processes across phase boundaries in miniplant-scale and continuous operation cannot be found to the best of our knowledge.…”

Three‐level hybrid modeling for systematic optimization of biocatalytic synthesis: α‐glucosyl glycerol production by enzymatic trans‐glycosylation from sucrose

“…Limitation on the applicability of mechanistic‐kinetic models can arise when their extension to the actual conditions of the biocatalytic synthesis proves difficult (Gonçalves et al, 2002 ; Grosch et al, 2017 ; Wandrey et al, 1979 ; Zhang et al, 2019 ). Although bulk conditions (e.g., pH, temperature, ionic strength, and enzyme preparation) may be invariant in the course of development, the high substrate concentrations of the synthetic process are typically avoided in studies of the kinetic mechanism, bearing in mind the possible convolution of specific and nonspecific effects of substrate used at high concentration.…”