Novel Glutamate Decarboxylase from Ethnic Food Metagenome: A Potential Biocatalyst to Produce a Nonproteinaceous Amino Acid, γ-Aminobutyric Acid

This study reports the biochemical characterization of a novel variant of glutamate decarboxylase (mtGAD), identified from an ethnic food, Kinema. The _<i>mt</i><i>gad</i> gene was cloned from the Kinema metagenome and expressed in a heterologous host, <i>Escherichia coli</i>. The enzyme mtGAD was extracted and purified for biochemical characterization. The enzyme showed optimal activity for γ-aminobutyric acid (GABA) synthesis at 50 °C and pH 4.5. The kinetic parameters of mtGAD were computed to be 29.2 mM <i>K</i>_m, 561.8 s^-1 <i>k</i>_cat, and 23.2 mM^-1 s^-1 <i>k</i>_cat/<i>K</i>_m. The <i>in silico</i> protein structure analysis, followed by molecular docking and molecular dynamics simulation, revealed that the binding with pyridoxal-5'-phosphate (PLP; a cofactor for mtGAD) reduces the fluctuations in the structure at high temperatures, contributing to the thermal stability of mtGAD. Furthermore, the conversion of about 60% monosodium glutamate (MSG) into GABA was achieved in approximately 3 h of catalytic reaction with 200 mM MSG, treated with 6.67 U mtGAD. A high turnover number with moderate thermal and pH stability makes mtGAD a potential biocatalyst for GABA production.