Molecular cloning, gene overexpression and characterization of glutamate decarboxylase from Enterococcus faecium DO

Abstract

In most bacteria, Glutamate decarboxylase (GAD) activity significantly reduces at pH > 6. Since l-glutamate is neutral to slightly alkaline, a large amount of hydrochloric acid must be added to the reaction mixture. Acidic reaction conditions greatly increase the corrosion of production equipment and shorten the life of manufacturing equipment. Therefore, this study aims at studying the overexpression of recombinant GAD that is active at the neutral pH and high-level production of γ-aminobutyric acid. A gene sequence encoding GAD of Enterococcus faecium DO was cloned into PET21a (+) and was overexpressed in Escherichia coli BL21 (DE3). The construct was confirmed by colony PCR and double digested with BamHI and XhoI. The result from Western blot analysis showed a signal band at the molecular mass of 54 kDa. The specific activity of the recombinant GAD was 78.17 U mg−1 at 45 °C and pH = 6.6. It was reduced rapidly when pH > 7.8. The Km, Vmax, and kcat values of recombinant GAD were 1.8 mM, 12.9 μmol L−1 min−1, and 0.43 s−1, respectively. Therefore, recombinant GAD is a potent candidate for GABA production in food and pharmaceutical industries, because it increases the efficiency of GABA synthesis, especially in foods with a neutral pH range.