International Institute for Carbon-Neutral Energy Research (WPI-I2CNER), Kyushu University, 744 Moto-oka, Nishi-ku, Fukuoka, 819-0395, Japan.
Extremophiles. 2014 Mar;18(2):363-73. doi: 10.1007/s00792-013-0621-x. Epub 2014 Jan 12.
Many types of superoxide dismutases have been purified and characterized from various bacteria, however, a psychrophilic Mn-superoxide dismutase (MnSOD) has not yet been reported. Here, we describe the purification and the biochemical characterization of the psychrophilic MnSOD from Exiguobacterium sp. strain OS-77 (EgMnSOD). According to 16S rRNA sequence analysis, a newly isolated bacterium strain OS-77 belongs to the genus Exiguobacterium. The optimum growth temperature of the strain OS-77 is 20 °C. The EgMnSOD is a homodimer of 23.5 kDa polypeptides determined by SDS-PAGE and gel filtration analysis. UV-Vis spectrum and ICP-MS analysis clearly indicated that the homogeneously purified enzyme contains only a Mn ion as a metal cofactor. The optimal reaction pH and temperature of the enzyme were pH 9.0 and 5 °C, respectively. Notably, the purified EgMnSOD was thermostable up to 45 °C and retained 50% activity after 21.2 min at 60 °C. The differential scanning calorimetry also indicated that the EgMnSOD is thermostable, exhibiting two protein denaturation peaks at 65 and 84 °C. The statistical analysis of amino acid sequence and composition of the EgMnSOD suggests that the enzyme retains psychrophilic characteristics.
已经从各种细菌中纯化和鉴定了许多类型的超氧化物歧化酶，但尚未报道过嗜冷锰超氧化物歧化酶（MnSOD）。在这里，我们描述了从极端嗜冷菌菌株 OS-77（EgMnSOD）中纯化和生化特性鉴定嗜冷 MnSOD 的过程。根据 16S rRNA 序列分析，新分离的细菌菌株 OS-77 属于极端嗜冷菌属。该菌株 OS-77 的最适生长温度为 20°C。EgMnSOD 是通过 SDS-PAGE 和凝胶过滤分析确定的 23.5 kDa 多肽的同源二聚体。紫外-可见光谱和 ICP-MS 分析清楚地表明，均相纯化的酶仅含有一个 Mn 离子作为金属辅因子。该酶的最佳反应 pH 和温度分别为 pH 9.0 和 5°C。值得注意的是，纯化的 EgMnSOD 在 45°C 下具有热稳定性，在 60°C 下孵育 21.2 分钟后保留 50%的活性。差示扫描量热法也表明，EgMnSOD 具有热稳定性，在 65 和 84°C 处表现出两个蛋白质变性峰。EgMnSOD 的氨基酸序列和组成的统计分析表明，该酶保留了嗜冷特性。
