The 11β-hydroxylation of 16,17α-epoxyprogesterone and the purification of the 11β-hydroxylase from Absidia coerulea IBL02
The 11β-hydroxylation product of 16,17α-epoxyprogesterone (EP), 11β-hydroxy-16,17α-epoxyprogesterone (HEP), is an important intermediate for many anti-inflammatory drugs. To understand the hydroxylation characteristics of the 11β-hydroxylase from filamentous fungus Absidia coerulea IBL02, the 11β-hydroxylation process, the purification results and some characteristics of the enzyme (protein P450) from mycelia were first reported in this paper. When the feeding time of the substrate was set at 27 h and the biotransformation time was for 42 h after fermentation optimization in the whole-cell bioconversion, a satisfied yield of about 4 g/l with above 85% biotransformation rate was achieved. We found the 11β-hydroxylase biosynthesis in cell cultivation could be induced by adding analogs of the substrate, and an about three-fold increase in enzymatic amount was achieved by adding progesterone as inducer for about 4 h. Experimental results showed that Ca2+ and Fe2+ could greatly enhance the enzymatic activity, but Mg2+, Mn2+, Cu2+, and Zn2+ could inhibit it. After the optimization of the purification conditions, the 11β-hydroxylase (protein P450) with a single band on SDS-PAGE was obtained and estimated to be a MW of about 60 kDa, giving a maximum absorbance at 448 nm in reduced carbon monoxide difference spectra.
Journal: Enzyme and Microbial Technology - Volume 41, Issues 1–2, 2 July 2007, Pages 71–79