Production of bleomycin N-acetyltransferase in Escherichia coli and Streptomyces verticillus
Open Access
- 1 August 1997
- journal article
- Published by Oxford University Press (OUP) in FEMS Microbiology Letters
- Vol. 153 (1) , 83-88
- https://doi.org/10.1016/s0378-1097(97)00237-1
Abstract
Bleomycin-producing Streptomyces verticillus ATCC 15003 has two bleomycin resistance genes, designated blmA and blmB . Bleomycin N- acetyltransferase, encoded by blmB , was overproduced in Escherichia coli 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 Xa protease, had an additional 6 N-terminal amino acid residues, but retained its bleomycin-acetylating activity, as did the entire fusion protein. The K m and V max 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 p I 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 -acetyltransferase is expressed simultaneously with bleomycin production in S. verticillus .Keywords
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