Isolation and Characterization of a Novel Thermostable DNA Polymerase from Bacillus Thermophilus A1  

Oleh:

Akhmaloka ; Nurbaiti, S. ; I. N. Tika ; Sindhumarta, M. ; Madayanti, F.

Jenis: Article from Journal - ilmiah nasional
Dalam koleksi: Jurnal Mikrobiologi Indonesia vol. 11 no. 2 (Sep. 2006), page 82-86.
Topik: isolation; thermostable DNA polymerase; bacillus thermophilus; ethylmaleimida; aphidicholin

Ketersediaan

Perpustakaan Pusat (Semanggi) Nomor Panggil: JJ139 Non-tandon: 1 (dapat dipinjam: 0) Tandon: tidak ada

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Isi artikel

Thermostable DNA polymerase (E.C.2.7.7.7) was isolated from local a thermophilic bacterium, bacillus thermophillus A1. Isolation was performed at mid logarithmic growth phase in a half recipe of luria bertani medium at 70 celcius degree. The crude enzyme was purified using ammonium sulfate fractionation, anion exchange, heparin sepharose and blue sepharose chromatography. DInally the active fraction from blue sepharose was subjected to sucrose gradient centrifugation. The activity of the enzyme was followed by incorporation of l(32) dATP on the growing chain of calf thymus DNA used as tempalte. The purified enzyme showed elevation in specific activity 220 times higher compared to that the crude enzyme, with purification yield at around 15,4%. However SDS-PAGE of the purified enzyme still showed two protein bands with the size around 89 and 70 kDa respectively. Characterization of the enzyme showed that the optimum activity was reached at pH 7,8 temperateure 80 celcius degree, Mg (2+), K (+) concentractions of 2,5 mM and 25mM respectively. At the p 6,8 or 8,8 the enzyme still showed 60% peack activity. Furthermore, at temperature 70 - 86 Celcius Degree the enzyme has remaining acticity at 50% of the maximal value. In addition, kinetic parameter analysis showed that Km for deoxy nucleotide triphosphate at 137 uM, Vmax = 1,152 U mg(-1) min (-1) and k (cat) = 15,94 U mg (-1) min (01). The activity of the enzyme was inhibited by aphidicholin and N ethylmaleimida with IC (50) 9,35 ug ml (-1) and 32,89 ug ml (-1) respectively. From all the data obtained suggest that the enzyme has different properties compared with other reported DNA polymerases from bacillus.

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