Molecular cloning and characterization of thermostable DNA ligase from Aquifex pyrophilus, a hyperthermophilic bacterium

Jae Hwan Lim, Juhyun Choi, Soo Jin Han, Sung Hou Kim, Hye Zin Hwang, Dong Kyu Jin, Byung Yoon Ahn, Ye Sun Han

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

A DNA ligase gene from the hyperthermophilic bacterium Aquifex pyrophilus (Ap) was cloned and sequenced. An open reading frame of 2,157 bp that codes for a 82-kDa protein showed 40%-60% homology with a series of NAD+-dependent DNA ligases from different organisms. The recombinant enzyme Ap DNA ligase expressed in Escherichia coli was purified to homogeneity and characterized. The activity of Ap DNA ligase gradually increased in proportion to the concentration of monovalent salt up to 200 mM NaCl, 150 mM KCl, 200 mM NH4Cl, and 350 mM potassium glutamate. The optimum temperature and pH of Ap DNA ligase were greater than 65°C and 8.0-8.6, respectively, for nick-closing activity. More than 75% of the ligation activity was retained after incubation at 95°C for 60 min, whereas the half-lives of Thermus aquaticus and Escherichia coli DNA ligases at 95°C were ≤15 min and 5 min, respectively. Thermostable Ap DNA ligase was applied to repeat expansion detection (RED) and could be a useful enzyme in DNA diagnostics.

Original languageEnglish
Pages (from-to)161-168
Number of pages8
JournalExtremophiles
Volume5
Issue number3
DOIs
Publication statusPublished - 2001

Keywords

  • Aquifex pyrophilus
  • DNA ligase
  • Hyperthermophile
  • Repeat expansion detection
  • Thermostability

ASJC Scopus subject areas

  • Microbiology
  • Molecular Medicine

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