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

Citation
Jh. Lim et al., Molecular cloning and characterization of thermostable DNA ligase from Aquifex pyrophilus, a hyperthermophilic bacterium, EXTREMOPHIL, 5(3), 2001, pp. 161-168
Citations number
35
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biotecnology & Applied Microbiology",Microbiology
Journal title
EXTREMOPHILES
ISSN journal
1431-0651 → ACNP
Volume
5
Issue
3
Year of publication
2001
Pages
161 - 168
Database
ISI
SICI code
1431-0651(200106)5:3<161:MCACOT>2.0.ZU;2-R
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(+)-depend ent DNA ligases from different organisms. The recombinant enzyme Ap DNA lig ase expressed in Escherichia coli was purified to homogeneity and character ized. The activity of Ap DNA ligase gradually increased in proportion to th e concentration of monovalent salt up to 200 mM NaCl, 150 mM KCl, 200 mM NH 4Cl, and 350 mM potassium glutamate. The optimum temperature and pH of Ap D NA ligase were greater than 65 degreesC and 8.0-8.6, respectively, for nick -closing activity. More than 75% of the ligation activity was retained afte r incubation at 95 degreesC for 60 min, whereas the half-lives of Thermus a quaticus and Escherichia coli DNA ligases at 95 degreesC were less than or equal to 15 min and 5 min, respectively. Thermostable Ap DNA ligase was app lied to repeat expansion detection (RED) and could be a useful enzyme in DN A diagnostics.