Immobilized glutamate decarboxylase by nano-CaCO 3 assisted alginate entrapment for γ-aminobutyric acid production

Glutamate decarboxylase (GAD) catalyzes the conversion of L-glutamate to γ-aminobutyric acid (GABA), a bioactive compound of therapeutic relevance. In this study, GAD was recombinantly expressed in Escherichia coli BL21 (DE3) (pET30a-GAD) and immobilized via entrapment in alginate gel with nano-calcium carbonate (nano-CaCO3) synthesized in a green deep eutectic solvent. The entrapment immobilization enhanced the nano-CaCO3 immobilized GAD catalytic properties, operational stability, and reusability. The immobilized GAD displayed optimal activity at 50 °C and pH 4, retained 42% activity after 3 h at 80 °C, and maintained 85% activity after 1 h at pH 3. The non-nano-CaCO3 immobilized GAD retained 21% activity after 3 h at 80 °C and 58% after 1 h at pH 3. The Km and Vmax of free GAD were 0.03 mM and 765.1 µmol·L−1·min−1. After immobilization, the Km and Vmax of non-nano-CaCO3 were 2.85 mM and 0.30 µmol⋅L−1 min−1, and the Km and Vmax of nano-CaCO3 were 1.37 mM and 0.48 µmol·L−1·min−1 respectively. Long-term storage and reusability assessments showed 52% retained activity after 40 days at 4 °C and over 90% after 8 cycles, compared to 27% and under 70% for the non-nano-CaCO3 immobilized GAD. The scanning electron microscopy (SEM) image confirmed the successful incorporation of nano-CaCO3 particles within the immobilized GAD beads. These results demonstrate that the nano-CaCO3 assisted alginate entrapment immobilization offers a robust and sustainable strategy for enhancing GAD stability and functionality in biocatalytic applications.