Inactivation of Blasticidin-S by Bacillus cereus. V. Purification and Characterization of Blasticidin S-Deaminase Mediated by a Plasmid from Blasticidin S Resistant Bacillus cereus K55-S1

摘要

Blasticidin S (BS) deaminase (BSR) from a BS-resistant strain, Bacillus cereus K55-S1, was purified to homogeneity. Molecular weights determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by gel filtration on HPLC are about 15500 and 35000, respectively, indicating the enzyme is a homodimer. The amino acid composition and N-terminal sequence of BSR are the same as those deduced from the nucleotide sequence of the BS-resistant gene, hsr. The optimum temperature and pH for enzyme activity are 60—65 °C and near 10.0, respectively. The activity of BSR is inhibited by Cu2+, Hg2 + , and/i-chloromercuric benzoate (PCMB). Inhibition by PCMB or HgCl2 is reversible by the addition of SH reagents. The enzyme catalyzes the deamination of BS and its derivatives, but not cytosine nucleosides.