Biotransformation of antibiotics

Abstract

The absence of a lag period in the bioconversion of chloramphenicol by spores and the pronounced influence of the pH of the medium on this reaction strongly suggested that chloramphenicol acetyltransferase (CAT) is located at or near the surface of the spores of Streptomyces griseus. A two-hour exposure of spores to dilute solutions of β-mercaptoethanol or surfactants resulted in significant decrease in activity even in the presence of glucose as an energy source. However, the inclusion of any of the reagents in the reaction mixture neither influenced the conversion activity nor the spore viability. These treatments did not reveal any cryptic activity for CAT in the spores. In addition, more drastic treatment of the spores with ethylenediaminetetraacetate (EDTA) or with concentrated salt solutions did not reduce the activity nor significantly affect the spore viability. Considering the modes of action of β-mercaptoethanol and the surfactants, a combination of disulfide bridges and lipoprotein interactions may be responsible for the binding of CAT to the surface of the spore. Moreover, results of acid treatment of intact spores indicated that most of CAT activity, if not all, is located at the spore surface. Incorporation of 14C-acetate by cell-free extracts of Streptomyces griseus clearly showed that CAT selectively catalyzed the formation of chloramphenicol-3-acetate at an optimum pH of 6.5. The shape of the pH-activity curve in cell-free extracts is essentially identical to that of the enzyme in intact spores and is additional evidence that the enzyme is located at the spore surface.