Purification and Properties of Extracellular Adenine Deaminase from Nocardioides sp. J-257L

Nocardioides sp. J-275L이 생산하는 세포외 Adenine Deaminase의 정제 및 성질

The extracellular adenine deaminase from Nocardioides sp. J-275L was purified by the following techniques: ammonium sulfate fractionation, DEAE-Cellulose, DEAE-Sephadex A-50 column chromatography, and Sephacryl S-200 superfine gel filtration. The enzyme was partially purified about 3889.5-fold with about 5.2% yield by these procedures. The molecular weight of the enzyme was 39,000 by a calibrated Sephacryl S-200 superfine column chromatography. The enzyme was stable at pH 7.5 and up to $40^{\circ}C$. Glycerol was effective on the stabilization of the enzyme during storage. The optimum pH and temperature of the enzyme were around pH 7.5 and $40^{\circ}C$, respectively. The apparent Michaelis constant Km of the enzyme for adenine was $7.4\times 10^{-5}$M. The purine analogues, 6-chloropurine, 2,6-diaminopurine, 6-bromopurine, 4-aminopyrazolo [3.4-d]pyrimidine, and 8-azaadenine were substrates for the enzyme. 6-Dimethylaminopurine was a competitive inhibitor of the enzyme. The enzyme was inhibited by 1mM of $Cu^{2+}, Fe^{3+}, Pb^{2+}, Hg^{2+}$, and $Ag^{+}$, and 1mM of $\alpha$,$\alpha$'-dipyridyl, pentachlorophenol, and pCMB.

Nocardioides sp. J-275L이 생산하는 세포의 adenine deaminase를 황산 암모늄 분획 , DEAE- Cellulose column c chromatography, DEAE-Sephadex A-50 column chromatography 및 Sephacryl S-200 superfine gel filtration 등의 파 정에 의해 5.2%의 수율로서 3889.5배로 부분정제하였으며, 본 효소의 분자량은 Sephacry] S-200 superfine gel에 의해 39, O 000 부근으로 측정되었다. 본 효소의 안정 pH와 온도는 pH7.와 $40^{\circ}C$로 나타났으며, 15%의 glycerol이 효소를 안정화시키는데 효과적이었다. 효소의 활성 최적 pH와 온도는 pH 7. 5 $40^{\circ}C$였다. 효소의 ademne에 대한 Km 값은 $7.4\times 10^{-5}$M로 측정되었으며, 검토된 punne a analogue 중에서 6-chloropurine. 2,6-diaminopurine, 6-bromopurine, 4-aminopyrazolo[3,4-dJ pyridine, 8-azaadenine 이 기질로 이용되었다. 본 효소는 0.1mM의 $Cu^{2+}, Fe^{3+}, Pb^{2+}, Hg^{2+}, Ag^{+}$ 등에 의해 활성이 크게 저해되었으며, $Pb^{2+}, Hg^{2+}, Ag^{+}$에 의해 저해된 효소의 활성은 EDTA에 의해 회복되었다. 검토된 일반적인 효소 저해제 중에서 1mM의 $\alpha$,$\alpha$'-dipyridyl, pentachlorophenol, and pCMB 등에 의해 효소의 활성 이 크게 저해되는 것으로 나타났다.