Optimization of Culture Conditions and Analysis of Plasmid Stability of a Transformant Bacillus subtilis for Cytidine Deaminase Production

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.