CHLOROPEROXIDASE FROM STREPTOMYCES-LIVIDANS - ISOLATION AND CHARACTERIZATION OF THE ENZYME AND THE CORRESPONDING GENE

For the first time, a halogenating enzyme which is not known to produc e halogenated metabolites has been isolated from a bacterial strain. T he gene encoding the nonheme chloroperoxidase (CPO-L) from Streptomyce s lividans TK64 was cloned, and its gene product was characterized. S. lividans TK64 produced only very small amounts of the enzyme. After c loning of the gene into Streptomyces aureofaciens Tu24-88, the enzyme was overexpressed up to 3,000-fold. Based on the overexpression, a sim ple purification procedure using acid precipitation and hydrophobic in teraction chromatography was developed. Thus, 54 mg of homogeneous CPO -L could be obtained from 27 g (wet weight) of mycelium. The native en zyme has a molecular weight of 64,000 and consists of two identical su bunits. The enzyme does not exhibit an absorption peak in the Soret re gion of the optical spectrum. X-ray fluorescence spectroscopy revealed that the enzyme does not contain any metal ions in equimolar amounts. CPO-L showed cross-reaction with antibodies raised against the nonhem e chloroperoxidase from Pseudomonas pyrrocinia but not with antibodies raised against CPO-T from S. aureofaciens Tu24. CPO-L exhibits substr ate specificity only for chlorination, not for bromination. Therefore, monochlorodimedone is only brominated by CPO-L, whereas indole is bro minated and chlorinated. The functional chloroperoxidase gene was loca ted on a 1.9-kb salI DNA fragment. DNA sequence analysis revealed an o pen reading frame encoding a predicted polypeptide of 276 amino acids. The overall identity of the amino acid sequence to that of chloropero xidase from P. pyrrocinia was 71%, whereas that to bromoperoxidase BPO -A2 from S. aureofaciens ATCC 10762 was only 42%.