Purification and characterization of thermostable cytidine deaminase encoded by the Bacillus caldolyticus cdd gene

Abstract

A 565 bp DNA stretch coding for the Bacillus caldolyticus DSM405 cdd gene was cloned by PCR amplification and sequenced. Nucleotide sequence analysis to the cloned DNA stretch revealed an open reading frame of 396 bp encoding a deduced polypeptide of 132 amino acids with a molecular mass of 14,235 Da. Comparison of the deduced polypeptide of the B. caldolyticus cdd gene to that of the Bacillus subtilis cdd showed a 71% sequence homology. Thermostable cytidine deaminase expressed in Escherichia coli JF611/pCJH53 was purified through rapid procedures consisting of anionic-exchange column chromatography and FPLC. Finally purified enzyme showed 167.5 u/mg of specific activity, corresponding to about 94-fold purification with respect to the crude extract. Molecular mass of the monomer was estimated to be about 14-kDa by SDS-polyacrylamide gel electrophoresis (PAGE), and tetrameric configuration of the native enzyme was supposed by gel filteration. Optimal deamination occurred at 70∼75°C and pH 7.5–8.0, and the activity was inhibited by Fe 2+, Cu 2+, Hg 2+, and p-chloromercuribenzoic acid ( pCMB). The enzyme was thermostable and the apparent K m and V max values for the cytidine were 2.1 × 10 −3 M and 36.4 mM min −1mg −1, respectively.