• 한국미생물·생명공학회지
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• 제25권1호
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• pp.9-14
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• 1997

A bacterium, strain YK 391 producing extracellular cytosine deaminase, has been isolated from soil sample collected near Taegu City and identified. The strain YK 391 was observed to be a motile Gram-negative rod, and did not produced capsule nor spore. The bacterium produced acid from glucose and trehalose, not from arabinose. Esculine was nto hydrolyzed. The isolate could grow anaerobically at 37$\circ $C, but not at 4$\circ $C. Palmitoleic and palmitic acids comprised over 80% of the fatty acid composition of the strain. The strain. The strain YK 391 was identified as Chromobacterium violaceum YK 391 based on its morphological and physiolohical characteristics, and on the fatty acid composition. The extracellular cytosine deaminase produced by Chromobacterium violaceum YK 391 is believed to be unique because it was active not only on cytosine and 5-fluorocytosine but also on cytidine.

• KSBB Journal
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• 제13권6호
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• pp.669-674
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• 1998

This study was carried out particularly focusing on he antitumor effect in combination with 5-fluorocytosine(5-FC), antifungal agent, and extracellular cytosine deaminase from Chromobacterium violaceum YK 391 against U-937, K-562 and SNU-C4 cells. While the addition of 10$\mu\textrm{g}$/100 ${\mu}\ell$ of anticancer agent, 5-fluorouracil(5-FU), to U-937, K-562 and SNU-C4 caused the decrease of proliferation 90%, 75% and 93% respectively, the addition of 20 $\mu\textrm{g}$/100 ${\mu}\ell$ of the extracellular cytosine deaminase and 10 $\mu\textrm{g}$/100 ${\mu}\ell$ of antifungal agent 5-FC caused the decrease of proliferation 80%, 70% and 90%, respectively. These results, therefore, reveal that this enzyme has the similar clinical effect for considering of adjuvant antitumor effect. From the above results, the treatment of 5-FC and the cytosine deaminase was very effective and showed the possibility to remove side effects which easily occur by the treatment of 5-FU only. An extracellular cytosine deaminase.

• 미생물학회지
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• 제26권4호
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• pp.368-374
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• 1988

Bacillus polymyxa YL38-3이 생성하는 세포의 cytosine deaminase의 효소학적 ,성질을 검토하였다. 본 세포외 효소는 열 안정성이 높으며, 인산완충액(pH6.0)과 $30^{\circ}C$에서 효소활성이 최대를 나타냈다. 본 효소는 cytosine 뿐 아니라 5- fluorocytosine-을 기질로 하나, 5-methylcytosine은 촉매하지 않았다. 더우기 본 효소는 $Cd^{2-}$, $Hg^{2+}$의 중금속이온과 ImM p-chloromercuribenzoate에 의하여 완전히 실활되며, o-phenanthroline과 monoiodoacetate에 의하여 75% 저해되었다. 그러나 1mM 2-mercaptoethanol에 의하여 본 효소의 활성을 약 200% 이상 활성화시켰다.

• 미생물학회지
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• 제26권4호
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• pp.362-367
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• 1988

The strain YL 38-3, which was capable of producing extracellular cytosine deaminase, was isolated and taxonomically examined. The isolated strain was identified to be Bacillus polymyxa YL 38-3. The optimal conditions for the enzyme production from Bacillus polymyxa YL 38-3 were investigated. The enzyme production was reached maximum level in the medium containing 0.5% glucose, 0.2% beef extract, 0.5% NaCl and 0.1% $KH_{2}PO_{4}$ (pH 6.0). And the enzyme showed the highest activity when the strain YL 38-3 was cultivated at $35^{\circ}C$ for 24 gours under the initial pH 6.0. By the additions of peptone the extracellular enzyme production was inhibited, meanwhile the intracellular enzyme production was highly stimulated. It was, therefore, deduced that peptone was related to the secretion mechanism of the enzyme from this bacterial cell.

• 미생물학회지
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• 제22권4호
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• pp.257-263
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• 1984

토양시료로 부터 cytosine과 5-fluorocytosine에 특이성을 갖는 세포의 cytosine deaminase를 생산하는 균주 JH-13을 분리하였다. 분리균 JH-13을 분류학적으로 검색한 결과 Arthrobacter 속에 속하는 것으로 밝혀졌으며 Arthrobacter JH-13으로 명명하였다. 분리 균의 세 포외 cytosine deaminase 생성을 위한 최적배지의 조성은 peptone 0.5%, meat extract 0.5%, soluble starch 0.5%, KCl O.1%로, 배지의 초발 pH 는 8.0으로 설정하였다. 500 ml용량의 진탕 flask에 최적배지 100 ml를 넣고 110 Rev.${\times}6$cm stroke로 $30^{\circ}C$에서 진탕배양하였을 때 효소생산은 54시간 부근에서 최고에 달하였다.

• Journal of Microbiology and Biotechnology
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• 제8권6호
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• pp.581-587
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• 1998

Essential amino acids involved in the catalytic role of the extracellular cytosine deaminase from Chromobacterium violaceum YK 391 were determined by chemical modification studies. The enzyme activity required the reduced form of Fe (II) ion, since the enzyme was inhibited by ο-phenanthroline. The enzyme activity was completely inhibited by the chemical modifiers, such as p-chloromercuribenzoate (p-CMB), p-hydroxymercuribenzoate, and chloramine-T at 1 mM each. The enzyme activity was also markedly inhibited by pyridoxal-5'-phosphate, diethyl pyrocarbonate, and phenylmethylsulfonyl fluroride at 1 mM each. The inactivation of the enzyme activity with p-CMB was reversed by a high concentration of cytosine. Furthermore, the inactivation of the enzyme activity with p-CMB was also reactivated by 1 mM dithiothreitol, 1 mM 2-mercaptoethanol, 1 mM cysteine-HCI, 10% ethyl alcohol, and 10% methyl alcohol. These results suggested that cysteine and methionine residues might be located in or near the active site of the enzyme, while lysine, histidine, and serine residues might be indirectly involved in the enzyme activity.

• Journal of Microbiology and Biotechnology
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• 제9권2호
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• pp.173-178
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• 1999

The extracellular cytosine deaminase (EC 3.5.4.1) from Chromobacterium violaceum YK 391 was purified 264.7-fold with an overall yield of 14.3%. The enzyme was for the first time homogeneous by the criteria of polyacrylamide gel electrophoresis performed in the absence and in the presence of sodium dodecyl sulfate. The molecular weight of the purified enzyme was estimated to be about 156 kDa. The enzyme consisted of two identical subunits of approximate molecular weight 78 kDa. The isoelectric point of the enzyme was pH 5.55. The enzyme had a pH optimum of 7.5 and a temperature optimum of around 40 to $45^{\circ}C$. Besides cytosine, the enzyme deaminated 5-fluorocytosine, cytidine, 5-methylcytosine, and 6-azacytosine, but not 5-azacytosine. The extracellular cytosine deaminase is believed to be unique because it was active not only on cytosine but also on cytidine. The apparent $K_m$ values for cytosine, 5-fluorocytosine, cytidine, and 5-methylcytosine were determined to be 1.55 mM, 5.52 mM, 10.4 mM, and 67.2 mM, respectively. The enzyme activity was strongly inhibited by heavy metal ions such as $Fe^{2+},Pb^{2+},Cd^{2+},Zn^{2+}, Hg^{2+}, and Cu^{2+}$ at 1 mM, and completely by $\alpha,\alpha$'-dipyridyl, and $\rho$-chloromercuribenzoate at 1 mM, and weakly inhibited by 1mM ο-phenanthroline. The enzyme activity was not affected by various nucleosides and nucleotides.

• 한국미생물·생명공학회지
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• 제20권2호
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• pp.150-155
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• 1992

토양 분리 균주로부터 얻은 배양액을 20-80 황산암모늄으로 분획한 후 세포의 cytosine deaminase의 성질을 검토하였다. 이 효소는 cytosine과 5-fluorocytosine(5-FC)에 기질 특이성을 가짐으로서 각각 uracil과 5-fluorouracil(5-FU)로 전환을 촉매하였다. 효소안정성에 대한 온도와 보존기간은 tris-HC1 완충용액에서 검토한 결과 최적온도는 $50^{\circ}C$ 부근이하에서 90 이상의 잔존활성을, 보존시간은 4일정도에서 80 이상의 잔존활성을 유지하였다. 최대 pH 8.0과 $37^{\circ}C$의 온도에서 나타났다. 활성에 적당한 pH는 8.0~8.5였고, 온도는 37~$45^{\circ}C$의 범위였다.

• 미생물학회지
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• 제23권3호
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• pp.177-183
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• 1985

20-80%로 황산암모늄 분획한 효소액을 사용하여 Arthrobacter sp. JH-13균주가 생산하는 세포외 cytosme deaminase의 성질을 검토하였다. 검토한 기질 중, 본 효소는 cytosine과 5- f1uorocytosine을 기질로 이용하였으며, 효소의 활성에 대한 최적 pH는 8.0부근이었고, 최적 온도는 $40^{\circ}C$부근으로 나타났다. 본 효소는 0.2M의 p potassium phosphate 완충액 (pH 8.0) 보다 0.2M의 tris-HCl 완충액 (pH 8. 0) 에서 더욱 안정하였다. 온도에 대한 안정성을 검토한 결과. $50^{\circ}C$ 부근까지는 대체로 안정하였으나, $70^{\circ}C$에서는 완전히 활성을 잃었다. 또한 ImM의 $Fe^{3+},\;K^+\;Na^+$ 이온은 효소의 활성을 증가시켰으나 0.01mM의 $Co^{2+},\;Cu^{2+},\;Ni^{2+},\;Hg^{2+},\;Ag^{2+},\;Zn^{2+},\;Ba^{2+},\;Mg^{2+}$ 이온들은 효소의 활성올 강력하게 저해하였다. O.lmM의 p-mercuribenzoate, trichloroacetic acid, N-ethylmaleim mide등은 효소의 활성을 완전히 저해하였으며. O.lmM의 2-mercaptoethanol은 효소의 활성올 약간 증가시키는 것으로 나타났다.

• Journal of Microbiology and Biotechnology
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• 제14권6호
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• pp.1182-1189
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• 2004

Cytosine deaminase (cytosine aminohydrolase, EC 3.5.4.1) stoichiometrically catalyzes the hydrolytic deamination of cytosine and 5-fluorocytosine to uracil and 5-fluorouracil, respectively. The intracellular cytosine deaminase from Chromobacterium violaceum YK 391 was purified to apparent homogeneity with 272.9-fold purification with an overall yield of $13.8\%$. The enzyme consisted of dimeric polypeptides of 63 kDa, and the total molecular mass was calculated to be approximately 126 kDa. Besides cytosine, the enzyme deaminated 5-fluorocytosine, cytidine, 6-azacytosine, and 5-methylcytosine, but not 5-azacytosine. Optimum pH and temperature for the enzyme reaction were 7.5 and $30^{\circ}C$, respectively. The enzyme was stable at pH 6.0 to 8.0, and at 30T for a week. About $70\%$ of the enzyme activity was retained at $60^{\circ}C$ for 5 min. The apparent $K_{m}$ values for cytosine, 5-fluorocytosine, and 5-methylcytosine were calculated to be 0.38 mM, 0.87 mM, and 2.32 mM, respectively. The enzyme activity was strongly inhibited by 1 mM $Hg^{2+},\;Zn^{2+},\;Cu^{2+},\;Pb^{2+},\;and\;Fe^{3+}$, and by o-phenanthroline, $\alpha,\;{\alpha}'$-dipyridyl, p-choromercuribenzoate, N-bromosuccinimide, and cWoramine­T. In addition, the enzyme activity was strongly inhibited by I mM 2-thiouracil, and weakly inhibited by 2-thiocytosine, or 5-azacytosine. Finally, intracellular and extracellular cytosine deaminases from Chromobacterium violaceum YK 391 were found to have a different optimum temperature, apparent $K_{m}$ value, and molecular mass.