Purification and characterization of glutamate decarboxylase from Lactobacillus brevis IFO 12005
Purification and characterization of glutamate decarboxylase from Lactobacillus brevis IFO 12005
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DOI：
10.1271/bbb.61.1168
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发表时间：
1997-07-01
影响因子：
1.6
通讯作者：
Oda, K
中科院分区：
文献类型：
--
作者：
Ueno, Y;Hayakawa, K;Oda, K
谷氨酸脱羧酶（GAD）[EC 4.1.1.15]通过Sephadex G-100、DEAE-Sepharose CL-6 B和Mono Q层析从短乳杆菌IFO 12005的无细胞提取物中纯化。从90.2g湿细胞中获得约9 mg纯化的GAD。纯化后的蛋白在SDS-PAGE上显示单一的蛋白带。SDS-PAGE和Superdex 200凝胶过滤纯化的GAD分子量分别为60，000和120，000，表明L. brevis作为二聚体存在。纯化的GAD N-末端氨基酸序列为NH 2-Met-Asn-Lys-Asn-Asp-Gln-Glu-Gln-Thr-。GAD的最适pH值为4.2，最适温度为30 ℃。硫酸根离子的加入使GAD活性呈剂量依赖性增加。影响顺序为：硫酸铵>硫酸钠>硫酸镁，说明亚基间疏水相互作用的增强导致了GAD活性的提高。纯化的GAD仅与作为底物的L-谷氨酸反应，K-m、k（cat）和k（cat）/K-m值分别为9.3 mM、6.5 s（-1）和7 x 10（2）M-1 s（-1）。
Glutamate decarboxylase (GAD) [EC 4.1.1.15] was purified from a cell-free extract of Lactobacillus brevis IFO 12005 by chromatographies on Sephadex G-100, DEAE-Sepharose CL-6B, and Mono Q. About 9 mg of purified GAD was obtained from 90.2 g of wet cells. The purified preparation showed a single protein band on SDS-PAGE. The molecular weights of purified GAD by SDS-PAGE and gel filtration on Superdex 200 were 60,000 and 120,000, respectively, indicating that GAD from L. brevis exists as a dimer. The N-terminal amino acid sequence of the purified GAD was NH2-Met-Asn-Lys-Asn-Asp-Gln-Glu-Gln-Thr-. The optimum pH and temperature of GAD were at pH 4.2 and at 30 degrees C. The GAD activity was increased by the addition of sulfate ions in a dose-dependent manner. The order of effect was as follows: ammonium sulfate > sodium sulfate > magnesium sulfate, indicating that the increase of hydrophobic interaction between subunits causes the increase of GAD activity. The purified GAD reacted only with L-glutamic acid as a substrate and the K-m, k(cat), and k(cat)/K-m values were 9.3 mM, 6.5 s(-1), and 7 x 10(2) M-1 s(-1), respectively.