Journal
BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY
Volume 72, Issue 5, Pages 1299-1306Publisher
TAYLOR & FRANCIS LTD
DOI: 10.1271/bbb.70782
Keywords
ammonium sulfate; glutamate decarboxylase (GAD); gamma-aminobutyric acid (GABA); Lactobacillus brevis
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In this study, the glutamate decarboxylase (GAD) gene from Lactobacillus brevis IFO12005 (Biosci. Biotechnol Biochem., 61, 1168-1171 (1997)), was cloned and expressed. The deduced amino acid sequence showed 99.6% and 53.1% identity with GAD of L. brevis ATCC367 and L. lactis respectively. The His-tagged recombinant GAD showed an optimum pH of 4.5-5.0, and 54kDa on SDS-PAGE. The GAD activity and stability was significantly dependent on the ammonium sulfate concentration, as observed in authentic GAD. Gel filtration showed that the inactive form of the GAD was a dimer. In contrast, the ammonium sulfate-activated form was a tetramer. CD spectral analyses at pH 5.5 revealed that the structures of the tetramer and the dimer were similar. Treatment of the GAD with high concentrations of ammonium sulfate and subsequent dilution with sodium glutamate was essential for tetramer formation and its activation. Thus the biochemical properties of the GAD from L. brevis IFO12W were significantly different from those from other sources.
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