Interdisciplinary Program in Genetic Engineering, Seoul National University, Seoul 151-742, Korea.
J Microbiol Biotechnol. 2009 Dec;19(12):1650-5. doi: 10.4014/jmb.0904.04053.
The beta-glucuronidase (GUS) gene from Lactobacillus brevis RO1 was cloned and expressed in Escherichia coli GMS407. The GUS gene was composed of 1812 bp, encoding a 603-amino-acid protein belonging to the glycosyl hydrolase family 2 with three conserved domains. The amino acid similarity was higher than 70% with the beta-glucuronidases of various microorganisms, yet less than 58% with the beta-glucuronidase of L. gasseri ADH. Overexpression and purification of the GUS was performed in beta-glucuronidase-deficient E. coli GMS407. The purified GUS protein was 71 kDa and showed 1284 U/mg of specific activity at optimum condition of pH 5.0 and 37 degrees C. At 37 degrees C, the GUS remained stable for 80 min at pH values ranging from 5.0 to 8.0. The purified enzyme exhibited a half-life of 1 h at 60 degrees C and more than 2 h at 50 degrees C. When the purified GUS was applied to transform baicalin and wogonoside into their corresponding aglycones, 150 microM of baicalin and 125 microM of wogonoside were completely transformed into baicalein and wogonin, respectively, within 3 h.
短乳杆菌 RO1 的β-葡萄糖醛酸酶（GUS）基因在大肠杆菌 GMS407 中被克隆和表达。GUS 基因由 1812bp 组成，编码一个 603 个氨基酸的蛋白质，属于糖苷水解酶家族 2，具有三个保守结构域。该氨基酸与各种微生物的β-葡萄糖醛酸酶的相似性高于 70%，但与 L. gasseri ADH 的β-葡萄糖醛酸酶的相似性低于 58%。在β-葡萄糖醛酸酶缺陷型大肠杆菌 GMS407 中进行 GUS 的过表达和纯化。纯化的 GUS 蛋白为 71kDa，在最佳条件 pH5.0 和 37°C 下具有 1284U/mg 的比活性。在 37°C 下，该酶在 pH5.0 到 8.0 范围内稳定 80 分钟。纯化酶在 60°C 下半衰期为 1 小时，在 50°C 下超过 2 小时。当纯化的 GUS 被应用于将黄芩苷和白杨素转化为相应的苷元时，150μM 的黄芩苷和 125μM 的白杨素在 3 小时内分别完全转化为黄芩素和白杨素。
