• Proceedings of the Korean Society for Applied Microbiology Conference
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• 1986.12a
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• pp.512.2-512
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• 1986

A mutant of bacillus subtilis with a defective cdd gene encoding deoxycytidine-cytidine deaminase(EC 3.5.4.5.) has been characterized genetically. The genetic lesion causing the altered deoxycytidine-cytidine deaminase, cdd, was mapped at 225 min on the linkage map of B.subtilis by AR9 transduction Transductional analysis of the cdd region established the gene order as trp-lys-dnaE-cdd-aroD. The cdd gene was linked 72% with the aroD and 20% with the lys.

• Microbiology and Biotechnology Letters
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• v.16 no.6
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• pp.536-539
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• 1988

A mutant of Bacillus subtilis with a defective cdd gene encoding deoxycytidine-cytidine deaminase (EC 3.5.4.5) has been characterized genetically. The genetic lesion, cdd, causing the altered deoxycytidine-cytidine deaminase was mapped at 225 min on the linkage map of B. subtilis by AR9 transduction, Transductional analysis of the cdd region established the gene order in clockwise as trp-lys-cdd-aroD. The cdd gene was linked 72% with the aroD and 20% with the lys.

• Journal of the Korean Society of Food Science and Nutrition
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• v.28 no.1
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• pp.87-93
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• 1999

This study was carried out to investigate the effects of nucleic acid related compounds and metal ions on activities of cytidine deaminase from Bacillus subtilis ED 213. The purified cytidine deaminase was weakly inhibited by 1mM GMP, IMP and ATP, but not affected by other nucleic acid related compounds such as CMP and UDP. The apparent Km values for cytidine, deoxycytidine, 5 methylcy tidine, fluorodeoxycytidine, and 5 bromocytidine were calculated to be 6.6$\times$10-4M, 6.0$\times$10-4M, 0.9$\times$10-4M, 0.8$\times$10-4M, and 2.0$\times$10-3M, respectively. The cytidine deaminase was completely inhibited by 1mM Hg2＋, and mildly inhibited over 40% by metal ions such as Na＋ and Fe2＋. However the enzyme activity was activated more than 40% by 1mM Mg2＋.

• Microbiology and Biotechnology Letters
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• v.17 no.4
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• pp.334-342
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• 1989

The Bacillus stearothermophilus cdd gene encoding cytidine deaminase (cytidine/2'-deoxycytidine aminohydrolase; EC 3.5.4.5) was isolated through shot gun cloning by oomplementation of an E. coli cdd mutation. Primarily 3.0 kbp of the exogenote was cloned into the Pstl site of pBR322 (pJSC101). By subsequent deletion and subcloning from the insert of pJSC101 with cdd$^+$ and tetracycline resistancy, about 1.35 kbp of the EcoRI$_1$/PstI$_2$ fragment containing the cdd gene was isolated as pJSC201. The minicell experiment revealed a molecular mass of 33,000 dalton for polypeptide from the cloned DNA fragment complementing the cdd gene. From the lacZ fusion of 550 bp fragment of the EcoRI$_1$/AuaI as a putative promoter region, the transcription direction of the cdd gene on pJSC201 is from EcoRI towards the PstI sites, When the cdd gene was expressed in B. subtilis ED4O (cdd$^-$, pyr$^-$) by transformation with the E. coli-B. subtilis shuttle vector, the gene expression occured more efficiently than in E. coli and the gene appears to be stably maintained in B. subtitis as well as in E. coli.

• Korean Journal of Environmental Biology
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• v.21 no.1
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• pp.60-65
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• 2003

The cytidine deaminase was partialy purified with sephadex G-200 and the characteristics of the enzyme were clarified. The molecular mass of the plasmid-encoded protein was identified as about 30 kDa in a minicell system. The native enzyme was estimated to have the molecular mass of 60 kDa by gel filtration. This indicates that the native enzyme may exist as a dimer composed of two identical subunits. The enzyme was reasonably stable in the pH range of 6 to 9, and was labile under high temperature above $50^{\circ}C$. Mercaptoethanol, pCMB, mercury and copper were found to inhibit the enzyme activity. The cytidine analogues of bromo- and iodo-(deoxy)-cytidine were also found to inhibit the activity, while fluorodeoxycytidine and azacytidine were found to activate it. Deoxycytidine, cytidine, ara-C and Methyldeoxycytidine have excellent substrates for the enzyme.

• Microbiology and Biotechnology Letters
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• v.18 no.6
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• pp.640-646
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• 1990

We have cloned the cdd gene from E. coli C600 using (cdd-) as a host. From the sequenced promoter region of E=, coli cdd gene which has been determined by Valentin-Hansen P. (1985), we synthesized the 23 mer oligonucleotides corresponding to the transcription initiation region and used as a probe for cloning of the cdd gene by Southern blotting. The isolated fragments in the blotting were introduced to the colony hybridization after transforming it into the E. coli JF611 (cdd-, pyr double mutant), and we identified the hybridized band at 27 kb long. From the original insert of 27 kb fragment in theBamHI site of pBR322, the 5.3 kb fragment containing the cdd gene was isolated by subsequent deletion and subeloning. From the derived plasmid pTK509, further deletion and subcloning were performed and clarified that the cdd gene was located in the 2.1 kb of SaZI/DraI segment in the insert of pTK605. The polypeptide encoded by the cloned DNA was appeared to be a molecular mass of 33,000.

• Microbiology and Biotechnology Letters
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• v.16 no.6
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• pp.468-475
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• 1988

The cloned B. subtilis cdd gene encoding cytidine/2'deoxycytidine deaminase (EC 3.5.4.5) was expressed in the cdd deficient B. subtilis mutant ED40. The gene was isolted from the cdd complementing plasmid pSO21, and inserted into the EcoR1/Pvu1 sites of pGB215-110 ΔB, which is a temperature sensitivie E. coli-B. subtilis shuttle vector. In the transformed B. subtilis ED4O harboring the resulting plasmid pSO100, cdd was expressed at several hundred fold elevated levels, and the cytidine deaminase activity in E. coli containing pSO100 was twice the level in B. subtilis/pSO0100. The Km value for cytidine of the partially purified enzyme is 1.88$\times$10$^{-4}$M at pH 7.0 and the V$_{max}$ = 11.1 $\mu$mol/min/mg of protein. The enzyme was completely inhibited by 0.1M mercaptoethanol and HgCl$_2$. The inhibition by p-chrolomercurybenzoic acid showed a Ki = 5 uM. These results suggest that sulfhydryl reagents block an active site thiol group, and/or disturb the formation of the tetrameic holoenzyme.

• Journal of Microbiology and Biotechnology
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• v.1 no.2
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• pp.116-120
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• 1991

The transformant Bacillus subtilis ED213 carrying the pSO100 which cloned the cdd gene encoding cytidine deaminase (cytidine /2'-deoxycytidine aminohydrolase, EC 3.5.4.5, CDase) originated from wild type B. subtilis was cultivated in Spizizen minimal medium (SMM). To overcome poor expression of the cdd gene in SMM medium, the medium compositions and growth conditions were optimized. The optimized medium compositions and growth conditions were cytidine concentration of 80 mg/l, glycerol of 25 g/l, and $(NH_4)_2SO_4$ of 10 g/l, along with $37^{\circ}C$ and pH 7.0. The intracellular CDase production was increased 3 times from 1,000 unit/ml to 3,200 unit/ml, and extracellular CDase also increased from nearly undetectable amounts to 1,500 unit/ml. The cytidine concentration was signified as the most critical compositional factor for overproduction of CDase by increasing the cell density mainly in culture broth. The plasmids were more stable in cells that were grown in original SMM medium with stability of 90% compared to those grown in optimized SMM medium with stability of 80% after 48 hours cultivation. The most active amplification of plasmid was occurred in the logarithmic phase, which showed a value around four times higher than the initial copy number. In the exponential phase, the CDase production was closely related to the plasmid copy number along with the cell density. However, it was not accorded with cell density at the stationary phase.

• Korean Journal of Environmental Biology
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• v.21 no.1
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• pp.56-59
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• 2003

The Salmonella typhimurium cdd gene encoding cytidine deaminase (cyti-dine/2'-deoxycytidine aminohydrolase; EC 3.5.4.5.) was isolated through shotgun clon-ing by complementation of the E. coli odd mutation. By subsequent deletion and sub-cloning from the original 3.7 Kb of EcoRI insert (pSAMI), the precise region of the cdd structural gene is located around the BglII site in the middle part of 1.7 Kb of NruI/PvuI segment. The 1.7 Kb containing odd gene wag subcloned to the pUC18 vector and the nucleotide sequence of the cdd gene was determined. When the putative ribosorne-binding site (Shine-Dalgarno sequence) and initiation codon were predicted to be GAGG at the position 459 and ATG at the position 470, respectively, there was an open reading frame of 885 nucleotides, encoding an 294 amino acid protein. The cdd gene expression in E. coli JF611/pSAMI was amplified about 50 fold compared to that of the wild type. The cdd gene expression was maintained in the stationary phase after rea-ching the peak in the late logarithmic phase.

• Journal of Microbiology and Biotechnology
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• v.1 no.1
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• pp.22-30
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• 1991

In order to secrete the Bacillus subtilis cytidine deaminase (CDase, cytidine/2'-deoxycytidine deaminase) encoded by the B. subtilis cdd gene in E. coli by the aid of signal sequences, the cdd gene was fused in-frame to either amyE or penP signal sequences and the gene expression and CDase localization were examined. For the penP signal sequence::cdd fusion, the cdd gene with 9 amino acids truncated from the 5'-terminus was fused in-frame to the signal sequence, then the $cdd^{+}$ colonies were not occurred from the minimal plate by cdd complementation. The result suggests that 9 amino acids on the $NH_2-terminal$ of CDase have an essential function in the enzyme activity. The hybrid protein obtained by fused gene amyE signal sequence::cdd structural gene gave $cdd^{+}$ phenotype and about half of the total CDase activity was found to be secreted in the periplasm of E. coli transformant JF611/pSO202. The periplasmic CDase activity of JF611 harboring pSO52 containing the intact cdd gene was considerablely lower than that of the cells harboring pSO202 carrying the hybrid cdd gene. This suggests that the CDase was secreted to the periplasm through the cytoplasmic membrane by the aid of the amyE signal sequence in the E. coli transformant.