Purification, properties and cDNA cloning of glutamate decarboxylase in germinated faba bean (Vicia faba L.)

Gamma-aminobutyric acid (GABA) is a non-protein amino acid with bioactive functions in humans. In this work, glutamate decarboxylase (EC 4.1.1.15, GAD) which is key in the GABA bioformation was purified from 5-day germinated faba beans and characterized. A single band was observed at 58 kDa using sodium dodecyl sulphate gel electrophoresis. GAD optimal activity was at pH 6.0 at 40 °C with a Km value for glutamic acid (Glu) of 2.63 mM. The enzyme was inhibited significantly by Cu2+, Fe3+, Mg2+, Ba2+, aminoxyacetate, EGTA, Na2EDTA, l-cysteine and beta-mercaptoethanol; and activated at low Ca2+ 0.2 mM. Using RT-PCR, the GAD cDNA was sequenced which indicated 1787 bp long, containing a 1527 bp open reading frame (ORF) that encoded 509 amino-acid peptides with a calculated molecular weight of 57.74 kDa and a pI of 5.41 (GenBank accession number: JX444699).