Kan Guangfeng, Wen Hua, Wang Xiaofei, Zhou Ting, Shi Cuijuan
School of Marine Science and Technology, Harbin Institute of Technology at Weihai, Weihai, China.
J Basic Microbiol. 2017 Aug;57(8):680-690. doi: 10.1002/jobm.201700165. Epub 2017 Jun 22.
A novel superoxide dismutase gene from Antarctic yeast Rhodotorula mucilaginosa AN5 was cloned, sequenced, and then expressed in Escherichia coli. The R. mucilaginosa AN5 SOD (RmFeSOD) gene was 639 bp open reading frame in length, which encoded a protein of 212 amino acids with a deduced molecular mass of 23.5 kDa and a pI of 7.89. RmFeSOD was identified as iron SOD type with a natural status of homodimer. The recombinant RmFeSOD showed good pH stability in the pH 1.0-9.0 after 1 h incubation. Meanwhile, it was found to behave relatively high thermostability, and maintained more than 80% activity at 50 °C for 1 h. By addition of 1 mM metal ions, the enzyme activity increased by Zn , Cu , Mn , and Fe , and inhibited only by Mg . RmFeSOD showed relatively low tolerance to some compounds, such as PMSF, SDS, Tween-80, Triton X-100, DMSO, β-ME, and urea. However, DTT showed no inhibition to enzyme activity. Using copper stress experiment, the RmFeSOD recombinant E. coli exhibited better growth than non-recombinant bacteria, which revealed that RmFeSOD might play an important role in the adaptability of heavy metals.
从南极酵母粘红酵母AN5中克隆、测序了一个新的超氧化物歧化酶基因，然后在大肠杆菌中进行表达。粘红酵母AN5超氧化物歧化酶（RmFeSOD）基因的开放阅读框长度为639 bp，编码一个由212个氨基酸组成的蛋白质，推导分子量为23.5 kDa，等电点为7.89。RmFeSOD被鉴定为铁超氧化物歧化酶类型，天然状态为同二聚体。重组RmFeSOD在1 h孵育后，在pH 1.0 - 9.0范围内表现出良好的pH稳定性。同时，发现其具有较高的热稳定性，在50℃下保温1 h后仍保持80%以上的活性。添加1 mM金属离子后，酶活性因锌、铜、锰和铁而增加，仅被镁抑制。RmFeSOD对一些化合物，如苯甲基磺酰氟、十二烷基硫酸钠、吐温-80、曲拉通X-100、二甲基亚砜、β-巯基乙醇和尿素的耐受性相对较低。然而，二硫苏糖醇对酶活性没有抑制作用。通过铜胁迫实验，表达RmFeSOD的重组大肠杆菌比未重组的细菌生长得更好，这表明RmFeSOD可能在重金属适应性中发挥重要作用。
