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KMID : 0578319910010030281
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Molecules and Cells
1991 Volume.1 No. 3 p.281 ~ p.286
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Purification and Properties of Escherichia coli Cytidine Deaminase by Amplification of the cdd Gene
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Kwon, Taeg Kyu
Lee, Sang Yong/Kim, Jong Guk/song, Bang-Ho/Hong, soon Duck
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Abstract
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The Escherichia coli cytidine/2´-deoxycytidine deaminase encoded by the cdd gene was purified from sonic extract of the transformant E. coli JF611 harboring the pTK148 containing the E coli cdd gene [Kwon, T. K. et al. (1990) J. Kor. Appl. Microbiol. Bioeng. 1$ 640-6461. The cytidine deaminase was purified to homogeneity by stepwise chromatography in the DEAE-cellulose, Sephadex G-200 and G-100, and DEAE-sephadex A-50 columns. From the gel filtration, the native enzyme was found to have a molecular mass of 62 kDa. Since the molecular mass of the polypeptide encoded by the pTK148 in SDS-polyacrylamide gel electrophoresis of the purified enzyme is 33 kDa, which is identical with that of the minicell experiment in our previous result, the cytidine deaminase might be composed of two identical subunits as a dimer. The optimal temperature and pH were 30 C and 7.0. The various aminopyrimidine nucleosides were also deaminated by this cytidine deaminase including, in the order of reactivity, deoxycytidine, fluorodeoxycytidine, 5-methyldeoxycytidine and cytidine. Thiol compounds like p-chloromercuribenzoate did not affect the enzyme activity so much, while various heavy metals like mercury, copper, and ferric ions did inhibit the enzyme activity significantly. The K. value of the enzyme was 6.6 X 10´ M, and the activation energy was 2.98 kcal/mole as determined by Arrhenius plot.
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