Department of Biochemistry, Microbiology and Biotechnology, Yerevan State University, Yerevan, Armenia.
Department of Biological Sciences, University of Bergen, Bergen, Norway.
Microbiologyopen. 2021 Jan;10(1):e1149. doi: 10.1002/mbo3.1149. Epub 2021 Jan 7.
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 Mg 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.
几种来自不同来源的天然和工程化耐热 DNA 聚合酶被用作不同分子技术的有力工具，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 株 K1 的 DNA 聚合酶（TsK1）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分支，与常用的 DNA 聚合酶 I 酶不同，包括来自 Thermus aquaticus 和 Thermus thermophilus 的酶。该酶的最佳温度和 pH 值分别为 72-74°C 和 9.0，能够有效地扩增 2.5kb 的 DNA 产物。TsK1 DNA 聚合酶不需要额外的 K 离子，但需要 3-5mM 的 Mg 才能达到最佳活性。它在 80°C 下至少稳定 1 小时，在 88°C 和 95°C 下的半衰期分别为 30 和 15 分钟。对扩增产物突变频率的分析表明，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，TsK1 DNA 聚合酶在各种分子应用中可能很有用，包括高温 DNA 聚合。
