The gene for BPO-A1, one of two non-haem bromoperoxidases in the tetracycline and 7-chlorotetracycline producer ATCC 10762, was cloned in the positive selection vector pIJ699 and expressed in TK64. The cloned bromoperoxidase was over-produced up to 2800-fold by the TK64 transformant. By taking advantage of the over-production of BPO-A1 and the heat stability of the enzyme, a new and simple purification procedure was developed. Subcloning into the vector pIJ487 and screening of recombinants by a newly developed histochemical assay located the gene on a 2•1 kb HI-dIII fragment. The nucleotide sequence of the 2•1 kb fragment was determined; the gene was identified within the sequence on the basis of the biased codon usage of genes and the presence of a nucleotide sequence encoding the N-terminal amino acid sequence obtained from the purified BPO-A1. Comparison of the deduced primary structure of BPO-A1 with those deduced for the non-haem chloroperoxidase CPO-P from and the bromoperoxidase BPO-A2 from ATCC 10762 gave amino acid sequence identities of 49% and 40%, respectively.


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