LAUSR

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 Km and Vmax 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.