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KMID : 1094720070120060707
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Biotechnology and Bioprocess Engineering
2007 Volume.12 No. 6 p.707 ~ p.712
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Cloning and expression of a full-length glutamate decarboxylase gene from Lactobacillus brevis BH2
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Kim Se-Hee
Shin Bo-Hye
Kim Yeon-Hee
Nam Soo-Wan
Jeon Seong-Soo
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Abstract
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A bacterium (BH2) that was found to produce a large amount of γ-aminobutyric acid (GABA) was isolated fromKimchi, a traditional fermented food in Korea. Phylogenetic analysis based on the 16S rDNA sequence and biochemical studies indicated that BH2 belonged to the genusLactobacillus 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 thegad 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 fromL. brevis showed 97.5 and 82.7% identities to theL. brevis OPK-3 GAD andL. plantarum WCFS1 GAD, respectively. Thegad gene was expressed inEscherichia coli cells and the expression was confirmed by SDS-PAGE analysis and enzyme activity studies.
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KEYWORD
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Lactobacillus brevis, γ-aminobutyric acid (GABA), gutamate decarboxylase (GAD), RACE-PCR
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