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Keywords:

  • glucose oxidase;
  • galactose oxidase;
  • lag time;
  • kinetic mechanism;
  • sensitivity analysis;
  • mutagenesis

Abstract

Parametric sensitivity analysis of a proposed model mechanism can serve as a valuable diagnostic for studying the fundamental characteristics of an enzymatic reaction. As the first step toward understanding the kinetics of a series of newly engineered glucose-6-oxidases, the activity of which has never been found in nature, we have proposed a mechanistic kinetic model and performed a parametric sensitivity analysis. The mechanism of our model consists of two enzyme conformations, namely “less active” and “more active,” and is shown to be consistent with experimental observations of prolonged periods of enzyme induction. The extended lag phase phenomenon was found to be well described mechanistically by a slow rate of interconversion between the two enzyme conformations (relative to the rate of product formation), and this prediction was further supported by our experimental results. The proposed enzymatic model will serve as a blueprint with which to better understand the mechanistic behavior of newly generated glucose-6-oxidases. © 2011 American Institute of Chemical Engineers AIChE J, 58: 2303–2308, 2012