State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, No.1 West Beichen Road, Chaoyang District, Beijing 100101, China.
Enzyme Microb Technol. 2011 Jun 10;49(1):94-9. doi: 10.1016/j.enzmictec.2011.03.001. Epub 2011 Mar 21.
The gene bglU encoding a cold-adapted β-glucosidase (BglU) was cloned from Micrococcus antarcticus. Sequence analysis revealed that the bglU contained an open reading frame of 1419 bp and encoded a protein of 472 amino acid residues. Based on its putative catalytic domains, BglU was classified as a member of the glycosyl hydrolase family 1 (GH1). BglU possessed lower arginine content and Arg/(Arg+Lys) ratio than mesophilic GH1 β-glucosidases. Recombinant BglU was purified with Ni2+ affinity chromatography and subjected to enzymatic characterization. SDS-PAGE and native staining showed that it was a monomeric protein with an apparent molecular mass of 48 kDa. BglU was particularly thermolabile since its half-life time was only 30 min at 30°C and it exhibited maximal activity at 25°C and pH 6.5. Recombinant BglU could hydrolyze a wide range of aryl-β-glucosides and β-linked oligosaccharides with highest activity towards cellobiose and then p-nitrophenyl-β-d-glucopyranoside (pNPG). Under the optimal conditions with pNPG as substrate, the K(m) and k(cat) were 7 mmol/L and 7.85 × 103/s, respectively. This is the first report of cloning and characterization of a cold-adapted β-glucosidase belonging to GH1 from a psychrotolerant bacterium.
从南极微球菌中克隆了编码冷适应β-葡萄糖苷酶（BglU）的基因 bglU。序列分析表明，bglU 包含一个 1419bp 的开放阅读框，编码 472 个氨基酸残基的蛋白质。根据其假定的催化结构域，BglU 被归类为糖苷水解酶家族 1（GH1）的成员。BglU 的精氨酸含量和 Arg/(Arg+Lys) 比值均低于嗜温 GH1 β-葡萄糖苷酶。用 Ni2+亲和层析纯化重组 BglU 并进行酶学特性分析。SDS-PAGE 和 native 染色表明，它是一种单体蛋白，表观分子量为 48kDa。BglU 特别不稳定，因为它在 30°C 时半衰期仅为 30 分钟，在 25°C 和 pH6.5 时表现出最大活性。重组 BglU 可以水解广泛的芳基-β-葡萄糖苷和β-连接的低聚糖，对纤维二糖的活性最高，然后是对硝基苯基-β-D-葡萄糖苷（pNPG）。在以 pNPG 为底物的最佳条件下，K(m)和 k(cat)分别为 7mmol/L 和 7.85×103/s。这是首次从耐冷菌中克隆和表征属于 GH1 的冷适应β-葡萄糖苷酶。
