极端嗜热古菌——芝田硫化叶菌DNA解旋酶的分离纯化和性质研究

采用Qsepharose离子交换层析、磷酸纤维素P1 1吸附层析、肝素琼脂糖吸附层析、Su perdex 2 0 0凝胶过滤和PhenylSuperose疏水层析等步骤 ,从嗜酸热芝田硫化叶菌细胞裂解液中分离纯化了一个DNA解旋酶。该解旋酶具有受DNA激活的ATP酶活性。根据SDS PAGE测定结果 ,该酶的分子质量约为 63kD。芝田硫化叶菌DNA解旋酶可以解开底物上 70bp的双链区 ,其解旋活性依赖于双链区旁的单链分叉。该解旋酶的活性依赖于Mg2 + 和ATP的水解 ,在NaCl浓度超过 2 0 0mmol L时受到抑制。该酶的最适pH为 6 7。该酶在 40℃～ 80℃之间均有活性 ,70℃时活性最高。芝田硫化叶菌DNA解旋酶是从古菌中分离得到的第一个天然DNA解旋酶。

PURIFICATION AND CHARACTERIZATION OF A DNA HELICASE FROM THE HYPERTHERMOPHILIC ARCHAEON SULFOLOBUS SHIBATAE

A DNA helicase from the hyperthermophilic archaeon Sulfolobus shibatae has been purified to homogeneity by column chromatography on Q Seqharose, phosphocellulose P11, heparin agarose, Superdex 200 and phenyl Superose. The purified enzyme shows DNA-stimulated ATPase activity. The molecular mass of the enzyme is approximately 63 kD. The S. shibatae DNA helcase is capable of unwinding a 70 bp duplex DNA flanked by unpaired single-stranded tails at both ends. The helicase activity requires Mg2+ and hydrolysis of ATP, and is inhibited by NaCl at concentrations greater than 200 mmol/L. It has an optimal pH of 6.7. The enzyme is active at temperatures between 40 and 80 degrees C, and the activity peaks at 70 degrees C. The S. shibatae DNA helicase is the first native DNA helicase to be isolated from archaea.