Department of Bioscience and Biotechnology, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul, 143-701, Republic of Korea.
Appl Biochem Biotechnol. 2010 Apr;160(8):2236-47. doi: 10.1007/s12010-009-8705-x. Epub 2009 Jul 22.
We expressed a putative beta-galactosidase from Sulfolobus acidocaldarius in Escherichia coli and purified the recombinant enzyme using heat treatment and Hi-Trap ion-exchange chromatography. The resultant protein gave a single 57-kDa band by SDS-PAGE and had a specific activity of 58 U/mg. The native enzyme existed as a dimer with a molecular mass of 114 kDa by gel filtration. The maximum activity of this enzyme was observed at pH 5.5 and 90 degrees C. The half-lives of the enzyme at 70, 80, and 90 degrees C were 494, 60, and 0.2 h, respectively. The hydrolytic activity with p-nitrophenyl(pNP) substrates followed the order p-nitrophenyl-beta-D-fucopyranoside > pNP-beta-D-glucopyranoside > pNP-beta-D-galactopyranoside > pNP-beta-D-mannopyranoside > pNP-beta-D-xylopyranoside, but not toward aryl-alpha-glycosides or pNP-beta-L-arabinofuranoside. Thus, the enzyme was actually a beta-glycosidase. The beta-glycosidase exhibited transglycosylation activity with pNP-beta-D-galactopyranoside, pNP-beta-D-glucopyranoside, and pNP-beta-D-fucopyranoside in decreasing order of activity, in the reverse order of its hydrolytic activity. The hydrolytic activity was higher toward cellobiose than toward lactose, but the transglycosylation activity was lower with cellobiose than with lactose.
我们在大肠杆菌中表达了来自嗜酸热硫化叶菌的假定β-半乳糖苷酶，并通过热处理和 Hi-Trap 离子交换层析纯化了重组酶。所得蛋白质在 SDS-PAGE 中呈现单一的 57 kDa 条带，比活为 58 U/mg。通过凝胶过滤，天然酶以二聚体形式存在，分子量为 114 kDa。该酶的最大活性在 pH 5.5 和 90°C 下观察到。该酶在 70、80 和 90°C 下的半衰期分别为 494、60 和 0.2 h。对 p-硝基苯（pNP）底物的水解活性遵循以下顺序：p-硝基苯-β-D-岩藻吡喃糖苷＞pNP-β-D-葡萄糖吡喃糖苷＞pNP-β-D-半乳糖吡喃糖苷＞pNP-β-D-甘露吡喃糖苷＞pNP-β-D-木吡喃糖苷，但对芳基-α-糖苷或 pNP-β-L-阿拉伯呋喃糖苷没有活性。因此，该酶实际上是一种β-糖苷酶。β-糖苷酶对 pNP-β-D-半乳糖吡喃糖苷、pNP-β-D-葡萄糖吡喃糖苷和 pNP-β-D-岩藻吡喃糖苷具有转糖基化活性，活性顺序依次降低，与水解活性相反。该酶对纤维二糖的水解活性高于乳糖，但对纤维二糖的转糖基化活性低于乳糖。
