Several native and engineered heat‐stable DNA polymerases from a variety of sources are used as powerful tools in different molecular techniques, including polymerase chain reaction, medical diagnostics, DNA sequencing, biological diversity assessments, and in vitro mutagenesis. The DNA polymerase from the extreme thermophile, Thermus scotoductus strain K1, (TsK1) was expressed in Escherichia coli, purified, and characterized. This enzyme belongs to a distinct phylogenetic clade, different from the commonly used DNA polymerase I enzymes, including those from Thermus aquaticus and Thermus thermophilus. The enzyme demonstrated an optimal temperature and pH value of 72–74°C and 9.0, respectively, and could efficiently amplify 2.5 kb DNA products. TsK1 DNA polymerase did not require additional K⁺ ions but it did need Mg²⁺ at 3–5 mM for optimal activity. It was stable for at least 1 h at 80°C, and its half‐life at 88 and 95°C was 30 and 15 min, respectively. Analysis of the mutation frequency in the amplified products demonstrated that the base insertion fidelity for this enzyme was significantly better than that of Taq DNA polymerase. These results suggest that TsK1 DNA polymerase could be useful in various molecular applications, including high‐temperature DNA polymerization.

中文翻译：

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极端嗜热菌 Thermus scotoductus K1 菌株 DNA 聚合酶 I 的特征

来自各种来源的几种天然和工程化热稳定 DNA 聚合酶被用作不同分子技术的强大工具，包括聚合酶链式反应、医学诊断、DNA 测序、生物多样性评估和体外诱变。来自极端嗜热菌 Thermus scotoductus K1 ( Ts K1)的 DNA 聚合酶在大肠杆菌中表达、纯化和表征。这种酶属于一个独特的系统发育进化枝，不同于常用的 DNA 聚合酶 I 酶，包括来自水生栖热 菌和嗜 热栖热菌的酶. 该酶的最佳温度和 pH 值分别为 72–74°C 和 9.0，可有效扩增 2.5 kb DNA 产物。Ts K1 DNA 聚合酶不需要额外的 K ⁺离子，但它确实需要 3-5 mM 的 Mg ²⁺以获得最佳活性。它在 80°C 至少稳定 1 小时，在 88 和 95°C 的半衰期分别为 30 和 15 分钟。扩增产物中的突变频率分析表明，该酶的碱基插入保真度明显优于Taq DNA 聚合酶。这些结果表明Ts K1 DNA 聚合酶可用于各种分子应用，包括高温 DNA 聚合。