Cloning and expression of a full-length glutamate decarboxylase gene from Lactobacillus brevis BH2

A bacterium (BH2) that was found to produce a large amount of gamma-aminobutyric acid (GABA) was isolated from Kimchi, a traditional fermented food in Korea. Phylogenetic analysis based on the 16S rDNA sequence and biochemical studies indicated that BH2 belonged to the genus Lactobacillus brevis. Under controlled conditions in MRS broth (Difco) with 5% monosodium glutamate, this strain produced GABA at a concentration of 194 mM with a 73% GABA conversion rate after 48 h. A full-length glutamate decarboxylase (gad) gene was cloned by the rapid amplification of cDNA ends (RACE) PCR. The open reading frame (ORF) of the gad gene was composed of 1,407 nucleotides and encoded a protein (468 amino acids) with a predicted molecular weight of 53.5 kDa. The deduced amino acid sequence of GAD from L. brevis showed 97.5 and 82.7% identities to the L. brevis OPK-3 GAD and L. plantarum WCFS1 GAD, respectively. The gad gene was expressed in Escherichia coli cells and the expression was confirmed by SDS-PAGE analysis and enzyme activity studies. (C) KSBB.