Cloning of a second non-haem bromoperoxidase gene from Streptomyces aureofaciens ATCC 10762: sequence analysis, expression in Streptomyces lividans and enzyme purification.
The gene for BPO-A1, one of two non-haem bromoperoxidases in the tetracycline and 7-chlorotetracycline producer Streptomyces aureofaciens ATCC 10762, was cloned in the positive selection vector pIJ699 and expressed in Streptomyces lividans TK64. The cloned bromoperoxidase was over-produced up to 2800-fold by the S. lividans 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 bpoA1 gene on a 2.1 kb BamHI-HindIII fragment. The nucleotide sequence of the 2.1 kb fragment was determined; the bpoA1 gene was identified within the sequence on the basis of the biased codon usage of Streptomyces 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 Pseudomonas pyrrocinia and the bromoperoxidase BPO-A2 from S. aureofaciens ATCC 10762 gave amino acid sequence identities of 49% and 40%, respectively.
Institut für Industrielle Genetik, Universität Stuttgart, Germany.
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I
Pelletier
O
O
Pfeifer
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Altenbuchner
K H
KH
van Pée
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010544:Peroxidases / Q000235:genetics* / Q000302:isolation & purification; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000235:genetics; D013304:Streptomyces aureofaciens / Q000201:enzymology* / Q000235:genetics*
D004269:DNA, Bacterial; D010544:Peroxidases; C026195:bromide peroxidase