Cloning and Characterization of Recombinant Glutamate Decarboxylase from <i>Coffea arabica</i> Leaves: Structural Insights and Ultrasound-Induced Modifications

Gamma-aminobutyric acid (GABA), a nonprotein amino acid with diverse functions, is synthesized by glutamate decarboxylase (GAD). While ultrasound has been shown to affect GAD activity, the underlying mechanism remains unclear. In this study, we successfully engineered a recombinant<i>Escherichia coli</i> (<i>E. coli</i>) strain carrying the <i>Coffea arabica</i> GAD (<i>CaGAD</i>) gene for the first time and characterized the bioinformatics characteristics of CaGAD. We then investigated the effects of ultrasound on the activity and structure of CaGAD purified from the recombinant <i>E. coli</i>. The results showed that ultrasonication at 20/40 kHz frequency, 15 W/L power, 28 °C, and a duration of 10 min increased the recombinant CaGAD activity by 27% compared to the untreated enzyme. The kinetic and thermodynamic analyses revealed that ultrasound enhanced the catalytic activity of recombinant CaGAD by reducing its Michaelis-Menten constant and activation energy. Multispectral analysis revealed that ultrasonication changed the enzyme's secondary and tertiary structures, while atomic force microscopy and particle size results demonstrated that ultrasound enhanced substrate accessibility and increased exposure of the active site. These findings suggest that ultrasound boosts the CaGAD activity by inducing structural changes and improving protein-substrate interactions.