Open AccessProduction of bleomycin N ‐acetyltransferase in Escherichia coli and Streptomyces verticillus
Author(s) -
Matsuo Hiroaki,
Mochizuki Hiroshi,
Davies Julian,
Sugiyama Masanori
Publication year - 1997
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.1997.tb10467.x
Subject(s) - escherichia coli , bleomycin , microbiology and biotechnology , streptomyces , acetyltransferase , chemistry , biology , bacteria , biochemistry , genetics , gene , acetylation , chemotherapy
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 X a 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 .