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KMID : 0545120150250050696
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Journal of Microbiology and Biotechnology
2015 Volume.25 No. 5 p.696 ~ p.703
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Characterization of Glutamate Decarboxylase (GAD) from Lactobacillus sakei A156 Isolated from Jeot-gal
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Sa Hyun-Deok
Park Ji-Yeong
Jeong Seon-Ju
Lee Kang-Wook
Kim Jeong-Hwan
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Abstract
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A gamma-aminobutyric acid (GABA)-producing microorganism was isolated from jeot-gal (anchovy), a Korean fermented seafood. The isolate, A156, produced GABA profusely when incubated in MRS broth with monosodium glutamate (3% (w/v)) at 37°C for 48 h. A156 was identified as Lactobacillus sakei by 16S rRNA gene sequencing. The GABA conversion yield was 86% as determined by GABase enzyme assay. The gadB gene encoding glutamate decarboxylase (GAD) was cloned by PCR. gadC encoding a glutamate/GABA antiporter was located immediately upstream of gadB. The operon structure of gadCB was confirmed by RT-PCR. gadB was overexpressed in Escherichia coli BL21(DE3) and recombinant GAD was purified. The purified GAD was 54.4 kDa in size by SDS-PAGE. Maximum GAD activity was observed at pH 5.0 and 55°C and the activity was dependent on pyridoxal 5’-phosphate. The Km and Vmax of GAD were 0.045 mM and 0.011 mM/min, respectively, when glutamate was used as the substrate.
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KEYWORD
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GABA, Lactobacillus sakei, glutamate decarboxylase, jeot-gal
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