School of Biological Sciences, University of the Punjab, Lahore, Pakistan 54590.
School of Biological Sciences, University of the Punjab, Lahore, Pakistan 54590.
Int J Biol Macromol. 2019 Nov 1;140:1194-1201. doi: 10.1016/j.ijbiomac.2019.08.150. Epub 2019 Aug 19.
Exonucleases catalyze the hydrolysis of terminal phosphodiester bond in nucleic acid. They play important role in maintaining the integrity of DNA in eukaryotes, prokaryotes and archaea. Limited studies have been done on archaeal exonucleases. Here we report molecular cloning of TK1646, a putative exonuclease from the hyperthermophilic archaeon Thermococcus kodakarensis, and expression of the gene in Escherichia coli. Recombinant TK1646, produced in soluble and active form, was purified to apparent homogeneity. Characterization of the recombinant enzyme indicated that it was single strand specific 3'-5' exonuclease which cleaved the substrate DNA after every two nucleotides. It exhibited highest activity at 85-100 °C and pH 9.0. Unique property of TK1646 was its thermostability as it maintained its activity even at 100 °C with a half-life of 180 min. Recombinant TK1646 followed Michaelis-Menten kinetics and exhibited apparent K and V values of 33 ± 4 μM and 812 ± 48 nmol/min/mg, respectively. To the best of our knowledge this is the most thermostable single strand specific 3'-5' exonuclease characterized to date.
核酸外切酶催化核酸末端磷酸二酯键的水解。它们在真核生物、原核生物和古菌中维持 DNA 完整性方面发挥着重要作用。目前对古菌核酸外切酶的研究还很有限。在这里，我们报道了来自高温古菌 Thermococcus kodakarensis 的假定核酸外切酶 TK1646 的分子克隆以及该基因在大肠杆菌中的表达。以可溶和活性形式产生的重组 TK1646 被纯化至明显的均一性。重组酶的特性表明，它是一种单链特异性 3'-5'核酸外切酶，每隔两个核苷酸切割底物 DNA。它在 85-100°C 和 pH 9.0 时表现出最高的活性。TK1646 的独特性质是其热稳定性，因为它即使在 100°C 下仍能保持活性，半衰期为 180 分钟。重组 TK1646 遵循米氏动力学，表现出明显的 K 和 V 值，分别为 33±4μM 和 812±48nmol/min/mg。据我们所知，这是迄今为止鉴定出的最耐热的单链特异性 3'-5'核酸外切酶。
