“…Traditional methods for enzyme kinetics, such as fluorescence spectroscopy, or other, usually indirect experimental methods, face limitations regarding environmental versatility, cost, and operational complexity. These limitations are particularly pronounced when analyzing samples with varying optical properties or in complex matrices, restricting the comprehensive assessment and utilization of engineered enzymes [3,[9][10][11][12] Innovative methodologies are necessary to determine critical kinetics parameters, such as the maximum reaction rate (v max ), Michaelis constant (K m ), and turnover number (k cat ), due to discrepancies in their values [6,[13][14][15][16][17][18]. This study aimed to address the aforementioned limitations by utilizing electrochemical impedance spectroscopy (EIS) to determine the kinetic parameters of genetically engineered enteropeptidases.…”