从泡菜中分离的短乳杆菌 HYE1 谷氨酸脱羧酶的表达和特性。
Expression and characterization of glutamate decarboxylase from Lactobacillus brevis HYE1 isolated from kimchi.
机构信息
Department of Life Sciences, Graduate School of Incheon National University, Incheon, Republic of Korea.
Research Group of Gut Microbiome, Korea Food Research Institute, Wanju, Republic of Korea.
出版信息
World J Microbiol Biotechnol. 2018 Mar 2;34(3):44. doi: 10.1007/s11274-018-2427-6.
A putative gene (gadlbhye1) encoding glutamate decarboxylase (GAD) was cloned from Lactobacillus brevis HYE1 isolated from kimchi, a traditional Korean fermented vegetable. The amino acid sequences of GADLbHYE1 showed 48% homology with the GadA family and 99% identity with the GadB family from L. brevis. The cloned GADLbHYE1 was functionally expressed in Escherichia coli using inducible expression vectors. The expressed recombinant GADLbHYE1 was successfully purified by Ni-NTA affinity chromatography, and had a molecular mass of 54 kDa with optimal hydrolysis activity at 55 °C and pH 4.0. Its thermal stability was determined to be higher than that of other GADs from L. brevis, based on its melting temperature (75.18 °C). Kinetic parameters including K and V values for GADLbHYE1 were 4.99 mmol/L and 0.224 mmol/L/min, respectively. In addition, the production of gamma-aminobutyric acid in E. coli BL21 harboring gadlbhye1/pET28a was increased by adding pyridoxine as a cheaper coenzyme.
从泡菜（一种传统的韩国发酵蔬菜）中分离出的短乳杆菌 HYE1 中克隆出了一种谷氨酸脱羧酶（GAD）的假定基因（gadlbhye1）。GADLbHYE1 的氨基酸序列与 GadA 家族有 48%的同源性，与短乳杆菌的 GadB 家族有 99%的同一性。该克隆的 GADLbHYE1 可通过诱导表达载体在大肠杆菌中功能性表达。表达的重组 GADLbHYE1 可通过 Ni-NTA 亲和层析成功纯化，其分子量为 54 kDa，最适水解活性为 55°C 和 pH 4.0。根据其熔点（75.18°C），其热稳定性被确定高于其他来自短乳杆菌的 GAD。动力学参数包括 GADLbHYE1 的 K 和 V 值，分别为 4.99 mmol/L 和 0.224 mmol/L/min。此外，在携带 gadlbhye1/pET28a 的大肠杆菌 BL21 中添加便宜的辅酶吡哆醇可提高 γ-氨基丁酸的产量。
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