STUDIES ON THE PREPARATION OF CELL-FREE PROGESTERONE HYDROXYLASES FROM ASPERGILLUS NIGER 12Y

Abstract

Preparation of cell-free progesterone hydroxylases from Aspergillus niger 12Y was achieved by extraction of the mycelium with acetate, citrate-phosphate, and phosphate buffer solutions at different pH values. All buffer solutions afforded cell-free preparations containing 11α-hydroxylase, 11β-hydroxylase, and 21-hydroxylase. The results indicated that 11α-hydroxylase and 21-hydroxylase comprised the major and minor components of the enzyme preparations, respectively. The extractability and/or the activity of the hydroxylases decreased and increased with the increase of the pH of acetate and citrate-phosphate buffers used in the mycelium extraction, respectively. Citrate-phosphate buffer provided enzyme preparations which were more active than those of acetate buffer. The latter buffer had a harmful effect on both the isolated hydroxylases and those remaining in the mycelium debris. On the other hand, grinding the mycelium with phosphate buffer at pH 6.24 provided the most active enzyme preparation. Homogenization of the mycelium with the last-mentioned buffer solution had an adverse effect on the activity of the resulting cell-free preparation. Addition of EDTA to the phosphate buffer of pH 6.24 resulted in enzyme preparations possessing weak 11α-hydroxylase and 21-hydroxylase activities and almost no 11β-hydroxylase activity.