THE PRODUCTION, ISOLATION, AND CHARACTERIZATION OF THE CHLOROPEROXIDASE ENZYME FROM Saccharothrix aerocolonigenes

Abstract

Rebeccamycin, a promising new antitumor antibiotic discovered by the Bristol-Myers Squibb Co., is isolated from fermentation of the actinomycete Saccharothrix aerocolonigenes. A water-soluble derivative of rebeccamycin is currently undergoing Phase I clinical trials as an anticancer agent. Although not proven, an indigenous chloroperoxidase is suspected of being involved in the biosynthesis of this antibiotic. The isolation and characterization of the enzyme responsible for halogenation of rebeccamycin was undertaken to provide Bristol-Myers Squibb with an important tool for production of halogenated analogues of novel antibiotics as anti-cancer agents. During this study a chloroperoxidase enzyme was isolated from a rebeccamycin-producing fermentation culture of S. aerocolonigenes. The enzyme was purified by fractional ammonium sulfate precipitation, diafiltration, and ion-exchange chromatography. Using monochlorodimedon as a surrogate substrate, it was determined that whole fermentation broth enzyme titer was at least 4.83 U/mL (enzyme units/mL), with a 30:70 distribution of the enzyme observed in culture supernate and biomass respectively. The enzyme has a pI below pH 6.0, is unstable in the absence of ammonium sulfate and/or stabilizing proteins, is heat labile, and will catalyze reaction of monochlorodimedon to dichlorodimedon in up to a 20% concentration of dimethyl suit oxide. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis shows prominent bands having the mobility of 27-29 kDa and 38 kDa molecular weight species, attributes which differentiate this product from the reference standard chloroperoxidase derived from Caldariomyces fumago which shows heavy bands at 58 KDa and 97 KDa. Our electrophoresis data coupled with the observed instability and intolerance to heat and lyophilization indicate that the isolated chloroperoxidase is not structurally related to the chloroperoxidase derived from Caldariomyces fumago. The ability of the S. aerocolonigenes derived enzyme to chlorinate monochlorodimedon also suggests that this enzyme is not a member of the non-heme type classification of bacterial haloperoxidase. It is anticipated that the data derived from this study will assist with the continued progression of research toward development of a more thorough understanding of this enzyme, and ultimately in the development of less toxic and more effective chemotherapeutics for cancer treatment.