• Microbiology and Biotechnology Letters
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• v.25 no.1
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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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• v.13 no.6
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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.

• Korean Journal of Microbiology
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• v.26 no.4
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• pp.368-374
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• 1988

Enzymological proprties of an extracellular cytosine deaminase from Bacellus polymyxa YL 38-3 were investigated. The extracellular enzyme was very stable, and optimum pH and temperature for the enzyme activity were found to be near pH 6.0 in 0.2M potassium phosphate buffer and at $30^{\circ}C$, respectively. 5-Fluorocytosine was converyed to 5-fluorouracil by the enzyme, but 5-methylcytosine was not to thymine by it. The enzyme activity was completely inhibited by some heavy metal ion such as 1mM of $Cd^{2-}$ and $Hg^{2+}$, and by 1mM of p-chloromercuribenzoate, respectively. The enzyme activity was inactivated about 75% by 1mM of o-phenanthroline and monoiodoacetate. But the enzyme activity was stimulated up to 200% by 1mM of 2-mercaptoethanol.

• Korean Journal of Microbiology
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• v.26 no.4
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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.

• Korean Journal of Microbiology
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• v.22 no.4
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• pp.257-263
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• 1984

A strain producing an extracellular cytosine deaminase was isolated from soil samples. The enzyme obtained from the strain possessed the substrate specificity to both cytosine and 5-fluorocytosine. From the results of its morphological, cultural, physiological, and biochemical properties, the strain was thought to be the genus Arthrobacter. Therefore, it was named as Arthrobacter sp. JH-13. The composition of optimum medium for the enzyme formation was 0.5% of peptone, 0.5% of meat extract, 0.5% of soluble starch, and 0.1% of KCl. The optimum pH for the enzyme formation was 8.0. When the microoganism was cultured aerobically in the above medium, enzyme production reached at maximum in 54 hours at $30^{\circ}C$.

• Journal of Microbiology and Biotechnology
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• v.8 no.6
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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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• v.9 no.2
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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.

• Microbiology and Biotechnology Letters
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• v.20 no.2
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• pp.150-155
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• 1992

- Some properties of an extracellular cytosine deaminase excreted from an isolate from soil samples were examined after 20~80%' ammonium sulfate fractionation. The enzyme catalyzed the conversion of cytosine and 5-fluorocytosine into uracil and 5-fluorouracil by substrate specificity, respectively. The optimum temperature and storage time on the stability of the enzvme preparation were below $50^{\circ}C$ keeping above 90% of the residual activity and near 4 days keeping above 80% of the residual one in Tris-HCI buffer. The maximum activity was also obtained at 8.0 in pH and 37'C in temperature. The pHs and temperatures for enzyme activity ranged from 8.0~8.5 and from 37~$45^{\circ}C$. respectively. The presence of $Ag^-,Hg^{2-}, Zn^{2-}, Cu^{2-}, Sn^{2-}, \; or\; Pb^{2-}$ in the reaction mixture resulted in the marked inhibition in enzyme activity, but 1 mM of $K^+, Fe^[3+}, Mg^{2+}, \; or \; Na^+$. slightly increased the activity. The enzyme preparation was heavily affected by most of inhibitors tested such as 1 mM of EIITA, NaCN and pentachlorophenol, and completely inactivated by p-CMB and TCA of 1 mM, or 10 mM.

• Korean Journal of Microbiology
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• v.23 no.3
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• pp.177-183
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• 1985

Some properties of an extracellular cytosine deaminase produced from Arthrobacter sp.JH-13 were examined after 20-80% of ammonium sulfate fractionation. Among some substrates, this enzyme utilized cytosine and 5-fluorocytosine as a substrate. The optimum pH and temperature for the activity of this enzyme were found to be near 8.0 and $40^{\circ}C$, respectively. The ensyme was more stable in 0.2M of Tris-HCl buffer than 0.2M of potassium phosphate buffer. The enzyme was generally stable below $50^{\circ}C$, but inactivated completely at $70^{\circ}C$. 1mM of $Fe^{3+},\;K^+\;and\;Na^+$ increased the enzyme activity, but 0.01mM of $Co^{2+},\;Cu^{2+},\;Ni^{2+},\;Hg^{2+},\;Ag^{2+},\;Zn^{2+},\;Ba^{2+},\;and\;Mg^{2+}$ markedly inactivated the enzyme activity. 0.1mM of p-chloromercuribenzoate, trichloroacetic acid, and N-ethylmaleimide compleyely inhibited the enzyme activity, but 0.1mM of 2-mercaptoethanol slightly increased the enzyme activity.

• Journal of Microbiology and Biotechnology
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• v.14 no.6
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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.