Creation of an Engineered Oxygen-Insensitive L-Glutamate Oxidase for the Application of Electrochemical L-Glutamate Sensors

L-glutamate (L-Glu) is the primary excitatory neurotransmitter in the mammalian central nervous system. Developing a real-time monitoring system is essential to understanding the onset and progression of related conditions. However, the absence of an L-Glu dehydrogenase that is insensitive to oxygen limits the development of oxygen-independent electrochemical enzymatic sensors. Additionally, the most commonly used L-Glu-specific oxidase requires site-specific proteolytic post-translational modifications in specific host microorganisms, which makes protein engineering difficult. To address these issues, L-Glu oxidase derived from <i>Streptomyces mobaraensis</i> (SmEOx), which does not require post-translational modifications, was engineered to function as a dehydrogenase. Residues crucial for the oxidative half reaction with oxygen in SmEOx were identified, and mutagenesis studies were conducted. Mutant SmEOx variants with suppressed oxidase activity and improved dye-mediated dehydrogenase activity compared to the wild-type enzyme were successfully obtained. The ratio of dehydrogenase activity to oxidase activity (Dh/Ox) increased ~2900-fold in mutant M117I and ~6700-fold in mutant M117F/K400N compared to wild-type recombinant SmEOx. The resulting virtually L-Glu dehydrogenases (<i>v</i>EDHs) were modified with a redox mediator and evaluated using transient open-circuit potential (OCP)-based L-Glu measurements. As a result, the <i>v</i>EDH (M117F/K400N mutant)-immobilized electrode enabled electrochemical L-Glu detection under ambient oxygen without the need for an external electron mediator, unlike the wild-type enzyme. The created <i>v</i>EDH, together with the OCP sensor developed using it, paves the way for future development of miniaturized, real-time L-Glu monitoring systems with high temporal and spatial resolution.