Purification and partial characterization of a thermostable ADN polymerase from the hyperthermophilic archaebacterium Thermococcus fumicolans
Raffin, J.P.; Dietrich, J. (1997). Purification and partial characterization of a thermostable ADN polymerase from the hyperthermophilic archaebacterium Thermococcus fumicolans, in: Biologie des sources hydrothermales profondes = Biology of deep-sea hydrothermal vents: Journées d'échanges du Programme DORSALES = DORSALES Workshop Roscoff 6-8 octobre 1997. Cahiers de Biologie Marine, 38(2): pp. 136
In: (1997). Biologie des sources hydrothermales profondes = Biology of deep-sea hydrothermal vents: Journées d'échanges du Programme DORSALES = DORSALES Workshop Roscoff 6-8 octobre 1997. Cahiers de Biologie Marine, 38(2)[s.n.][s.l.]. 111-149 pp., more
In: Cahiers de Biologie Marine. Station Biologique de Roscoff: Paris. ISSN 0007-9723; e-ISSN 2262-3094, more
|  |
| Authors | | Top |
- Raffin, J.P.
- Dietrich, J.
|
|
|
| Abstract |
The archaebacterium Thermococcus fumicolans was isolated from a deep-sea hydrothermal vent in the North Fiji Basin (Godfroy et al., 1996). The strain is an obligatory chemoorganotrophic, strictly anaerobic and grows preferentially in the presence of elemental sulphur. The optimal growth temperature is around 85 degree C. For the purpose of the study, T. fumicolans strain ST557 was grown in medium containing, per liter, 10 g of meat extract, 30 g of sea salt and 10 g of sulphur. The pH was adjusted, at room temperature, to 7.5. The medium was sterilized by two successive beatings at 100 degree C for 30 min and then reduced, before inoculation, by nitrogen bubbling and by adding 0.5 g l super(-1) of sodium sulphide. At the end of the log phase, the medium was allowed to cool and cells were harvested in a continuous flow centrifuge. Typically, about 400 g of cells were obtained in a total volume of 180 l. Cells were then frozen in liquid nitrogen and stored at -80 degree C. Cells were lysed by incubation at 37 degree C for 2 h in Tris buffer containing 1 g l super(-1) of lysozyme. After centrifugation, DNA had to be removed in order to get reproducible results on chromatographic supports. Therefore, the NaCl in the extract was adjusted to 1 M and DNA was precipitated by adding 0.37% (w/v) of Polymin P. The extract was then treated with ammonium sulphate to remove excess of Polymin and purification was performed by using Cellulose-phosphate, Phenyl-sepharose, Resource S, Affi-gel Heparin and Mono S chromatography. The enzyme was characterized by determination of kinetic parameters, thermal stability, processivity and exonuclease activity. The enzyme displayed a 3'-5'-exonuclease proof-reading activity. |
|
