Purification and Molecular and Catalytic Properties of Bromoperoxidase from Streptomyces phaeochromogenes

Journal of general microbiology 09/1985; 131(8):1911-6. DOI: 10.1099/00221287-131-8-1911
Source: PubMed

ABSTRACT A bromoperoxidase has been isolated and purified from the chloramphenicol-producing actinomycete Streptomyces phaeochromogenes. The purified enzyme was homogeneous as determined by polyacrylamide gel electrophoresis. The prosthetic group of the bromoperoxidase was ferriprotoporphyrin IX. Based on gel filtration results the molecular weight of the enzyme was 147 000 +/- 3000. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed a single band having the mobility of a 72 500 molecular weight species. Therefore, in solution at neutral pH, the bromoperoxidase behaved as a dimer. The isoelectric point was 4.0. The spectral properties of the native and reduced enzyme are reported. The homogeneous enzyme also had peroxidase and catalase activity.

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    ABSTRACT: 1.1. Extracts of a 25,000 g sediment of Murex trunculus hypobranchial gland homogenate contain an enzyme which brominates monochlorodimedon in the presence of bromide and H2O2.2.2. Dependence of activity on H2O2 concentration exhibits a sharp optimum which increases from 20 μM at pH 7.4 to 400 μM at pH Dependence on Br− concentration follows a simple saturation curve with Km values of 7 mM at pH 7.4 and 30 mM at pH The pH optima depend on H2O2 concentration, ranging from pH 6.6 at 10 μM to pH 4.8 at 200 μM.5.5. The enzyme is totally inactive with Cl− and F−, although both these ions inhibit bromination.
    Comparative biochemistry and physiology. B, Comparative biochemistry 01/1987; 88(3-88):917-922. DOI:10.1016/0305-0491(87)90264-1 · 2.07 Impact Factor
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    ABSTRACT: A bromoperoxidase gene was cloned from Streptomyces aureofaciens Tü24 into Streptomyces lividans TK64 by using the promoter-probe vector pIJ486. Subcloning of DNA from the original, unstable clone allowed the gene to be localized to a 1.7-kilobase (kb) fragment of DNA. Southern blotting showed that the cloned 1.7-kb insert hybridized to a 4.3-kb fragment in an SstI digest of S. aureofaciens Tü24 total DNA. The 1.7-kb insert was shown to code for a protein with the electrophoretic properties of the subunits of the nonheme bromoperoxidase isolated from S. aureofaciens Tü24. The protein produced by S. lividans TK64 transformed with pHM621, which contained an 8.0-kb insert, was shown to be identical to the S. aureofaciens Tü24 bromoperoxidase in terms of its electrophoretic mobility on denaturing and nondenaturing polyacrylamide gels and its NH2-terminal amino acid sequence. The bromoperoxidase was overproduced (up to 180 times) by S. lividans TK64 containing pHM621. Based on the heat stability of the S. aureofaciens Tü24 bromoperoxidase, a new and simple purification procedure with very high yields was developed.
    Journal of Bacteriology 01/1989; 170(12):5890-4. · 2.81 Impact Factor
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    ABSTRACT: A new bromoperoxidase-catalase was purified from the chloramphenicol-producing actinomycete Streptomyces venezuelae ISP 5230. The homogeneous enzyme showed brominating activity, catalase activity and a very low peroxidase activity. The spectral properties and pH dependence of the catalase activity showed similarities to conventional catalases. In contrast to other haem-bromoperoxidases, the bromoperoxidase-catalase was stable when treated with an ethanol/chloroform mixture. Gel filtration gave an estimated Mr of 127,000-136,000. SDS-PAGE showed a single band corresponding in mobility to a species with an Mr of 61,000. The pI was estimated to be 4.5. The bromoperoxidase-catalase was not present in active form in a mutant of S. venezuelae ISP 5230, blocked in the chlorination step of chloramphenicol biosynthesis. However, an inactive species of the enzyme was detected in crude extracts of the mutant by using antibodies. From these results it is concluded that this bromoperoxidase participates in the chlorination step during chloramphenicol biosynthesis.
    Journal of general microbiology 10/1989; 135(9):2493-502. DOI:10.1099/00221287-135-9-2493
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