Sa Hyun Deok, Park Ji Yeong, Jeong Seon-Ju, Lee Kang Wook, Kim Jeong Hwan
Division of Applied Life Science (BK21 Plus), Graduate School, Gyeongsang National University, Jinju 660-701, Republic of Korea.
Institute of Agriculture and Life Science, Gyeongsang National University, Jinju 660-701, Republic of Korea.
J Microbiol Biotechnol. 2015 May;25(5):696-703. doi: 10.4014/jmb.1412.12075.
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.
从韩国发酵海产品腌鱼中分离出一株产γ-氨基丁酸（GABA）的微生物。分离株A156在含有3%（w/v）谷氨酸钠的MRS肉汤中于37°C培养48小时时大量产生GABA。通过16S rRNA基因测序将A156鉴定为清酒乳杆菌。通过GABase酶法测定，GABA转化率为86%。通过PCR克隆了编码谷氨酸脱羧酶（GAD）的gadB基因。编码谷氨酸/GABA反向转运体的gadC位于gadB的紧邻上游。通过RT-PCR证实了gadCB的操纵子结构。gadB在大肠杆菌BL21(DE3)中过表达，并纯化了重组GAD。通过SDS-PAGE分析，纯化的GAD大小为54.4 kDa。在pH 5.0和55°C时观察到GAD的最大活性，且该活性依赖于磷酸吡哆醛。以谷氨酸为底物时，GAD的Km和Vmax分别为0.045 mM和0.011 mM/分钟。
