Engineering of Gamma-Aminobutyric Acid (GABA) Oxidase Towards the Development of a Quasi-Direct Electron Transfer Type Electrochemical GABA Sensor

Gamma-aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the mammalian central nervous system, and abnormal GABA regulation is associated with neurological disorders including autism, schizophrenia, and Alzheimer’s disease. Current electrochemical GABA sensors often rely on multi-enzyme systems that lack ideal specificity and sensitivity, motivating the development of a simpler GABA-specific sensing platform. In this thesis, Clonostachys rosea GABA oxidase (CrGABAOx) was rationally engineered toward a quasi-direct electron transfer (quasi-DET) electrochemical GABA sensor using amine-reactive phenazine ethosulfate (arPES) as a covalently attached redox mediator. Lysine mutations were introduced near the FAD cofactor to improve electron transfer between the enzyme and electrode surface. Among the single mutants tested, Y200K showed the strongest electrochemical performance, producing approximately two-fold greater chronoamperometric and open-circuit potential responses compared with wild-type CrGABAOx and T86K, while retaining substantial enzymatic activity. Double mutants incorporating Y200K were also expressed and characterized, with Y200K/N105K and Y200K/G106K retaining promising activity for future electrochemical evaluation. Overall, these results demonstrate that rational lysine engineering of CrGABAOx is a viable strategy for improving quasi-DET-based GABA sensing, while also highlighting the need to balance electron transfer efficiency with enzyme activity, stability, and productivity.