Abstract 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 Ca 2+ and Fe 2+ could greatly enhance the enzymatic activity, but Mg 2+ , Mn 2+ , Cu 2+ , and Zn 2+ 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.

中文翻译：

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16,17α-环氧孕酮的 11β-羟基化和蓝蓝夜蛾 IBL02 中 11β-羟化酶的纯化

摘要 16,17α-环氧孕酮（EP）的11β-羟基化产物11β-羟基-16,17α-环氧孕酮（HEP）是许多抗炎药物的重要中间体。为了解丝状真菌蓝斑病菌IBL02 11β-羟化酶的羟基化特性，本文首次报道了11β-羟基化过程、菌丝体中酶（蛋白P450）的纯化结果和一些特性。当在全细胞生物转化中发酵优化后，将底物的补料时间设置为27小时，生物转化时间为42小时，达到了约4g/l的满意产量，生物转化率在85%以上。我们发现可以通过添加底物类似物来诱导细胞培养中的 11β-羟化酶生物合成，加入黄体酮诱导剂约 4 小时，酶量增加约 3 倍。实验结果表明，Ca 2+ 和Fe 2+ 可以大大增强酶活性，但Mg 2+ 、Mn 2+ 、Cu 2+ 和Zn 2+ 可以抑制酶活性。纯化条件优化后，得到 11β-羟化酶（蛋白 P450）在 SDS-PAGE 上具有单条带，估计分子量约为 60 kDa，在还原一氧化碳差谱中在 448 nm 处具有最大吸光度.