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The steroid 9alpha-hydroxylase activity has been detected in cytosol fraction, 100,00OXg supernatant of cell free extract of Mycobacterium fortuitum. The activity was not linear viith protein concentration in the assay suggesting 9alpha-hydroxylase is a multicomponent enzyme. The 9alpha-hydroxylase system was partially purified through fractional saturation of ammonium sulfate, strong anion exchange (Mono Q) column chromatography, gel filtration (Superose 12) column chromatography, and testosterone affinity gel chromatography. Ammonium sulfate 50~60% saturated fraction of the cytosol gave 9alpha-hydroxylase activity. For further purification, the half-saturated ammonium sulfate fraction was applied to Mono Q, Superose 12, or affinity gel column. The purification factors of 9alpha-hydroxylase containing fraction after Mono Q, Superose 12, and affinity gel chromatography was 13, 11, and 17 respectively.
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