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

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• 作者：Jan-Hendrik Grosch ; David Wagner ; Niklas Knaup ; Timm Keil and Antje C. Spieß
• 刊名：Biotechnology Progress
• 出版年：2017
• 出版时间：January/February 2017
• 年：2017
• 卷：33
• 期：1
• 页码：87-95
• 全文大小：424K
• ISSN：1520-6033

文摘

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%.