Production of bleomycin N-acetyltransferase in Escherichia coli and Streptomyces verticillus

Bleomycin-producing Streptomyces verticillus</it> ATCC 15003 has two bleomycin resistance genes, designated blmA</it> and blmB</it>. Bleomycin N-</it> acetyltransferase, encoded by blmB</it>, was overproduced in Escherichia coli</it> as a protein fused to the maltose-binding protein. The protein (fBAT), purified to homogeneity after digestion of the fusion product with blood coagulation factor X<inf>a</inf> protease, had an additional 6 N-terminal amino acid residues, but retained its bleomycin-acetylating activity, as did the entire fusion protein. The K</it><inf>m</inf> and V</it><inf>max</inf> values of purified fBAT for the substrate bleomycin were 13.0 μM and 3.4 pmol min−1 ml−1, respectively. The optimal pH for the acetylating activity was 6.0 in 10 mM phosphate buffer. The molecular mass and pI</it> value of fBAT were estimated by polyacrylamide gel electrophoresis to be about 34 500 and 6.13, respectively. An anti-fBAT monoclonal antibody was generated and used to show that bleomycin N</it>-acetyltransferase is expressed simultaneously with bleomycin production in S. verticillus</it>.