Transformation of progesterone by fungal systems : studies on the 11 -hydroxy;ase system from aspergillus ochraceus

Abstract

Transformation of Progesterone by Fungal Systems: Studies on the 11 Hydroxylase System from Aspergillus ochraceus.” Microbial hydroxylations of steroids have gained industrial importance since these reactions are commonly used in the synthesis of pharmacologically active steroid hormones, which are sometimes difficult to synthesize by conventional chemical routes. Fungi are often used to introduce hydroxyl groups into a steroid skeleton. Various fungi of the genera Aspergillus and Rhizopus have been shown to hydroxylate progesterone and related steroids at the 11 position. 11 Hydroxylation of progesterone is one of the industrially important fungal hydroxylations, and there are innumerable reports on this transformation. However, there are very few reports pertaining to the nature of the enzyme system involved in this specific hydroxylation. Efforts made by many investigators to characterize this hydroxylase did not yield conclusive information. Until recently, even the subcellular localization of this hydroxylase system had not been conclusively established. In the present study, the enzyme system of Aspergillus ochraceus involved in the conversion of progesterone to 11 hydroxyprogesterone has been studied in great detail. The optimal conditions for high hydroxylase activity with conidia and vegetative cells of A. ochraceus were established. The 11 hydroxylase system was found to be inducible. Among the different steroids tested for their ability to induce the 11 hydroxylase system, progesterone was found to be the most efficient inducer. Androstenedione and testosterone induced to a moderate extent, while deoxycorticosterone did not induce the activity. The induction of hepatic microsomal enzymes in animals treated with barbiturates is well known. However, phenobarbital did not induce the 11 hydroxylase system. The inducible nature of the hydroxylase activity was further supported by the inhibition of the hydroxylase activity by cycloheximide added during the induction period. Attempts to prepare cell free extracts from the spores and vegetative cells of A. ochraceus by various methods such as freezing and thawing, homogenization, acetone treatment, sonication of the spores and the preparation of protoplasts by treatment of the spores as well as mycelia with snail gut enzyme, glucuronidase and chitinase resulted in preparations devoid of any activity. Active cell free extracts were prepared by grinding the vegetative cells with glass powder in the presence of a suitable buffer. The ideal conditions to obtain maximum hydroxylase activity at the cell free level were standardized. The 11 hydroxylase activity was found to be solely localized in the microsomal fraction. This constitutes the first report on the intracellular localization of this steroid hydroxylase system from fungi.