赭曲霉甾体11α-羟化酶基因异源表达研究

发布时间：2018-05-01 22:53

本文选题：甾体11α-羟化酶 + 酿酒酵母　；参考：《天津科技大学》2017年硕士论文

【摘要】：赭曲霉在工业上用于催化16α,17α-环氧黄体酮C11α-羟化反应,合成重要糖皮质激素类药物中间体C11α-羟基-16α,17α-环氧黄体酮。参与甾体C11α-羟基化反应的赭曲霉羟化酶为细胞色素P450酶。本课题首先在酿酒酵母中异源表达C1 1α-羟化酶基因AOH,结果表明AOH重组酵母细胞能够转化16α,17α-环氧黄体酮形成产物C1 1α-16α,17α-环氧黄体酮,说明AOH基因在酵母细胞中实现了功能表达。由于赭曲甾体C11α-羟化酶在工业上的价值,分析该酶的结构与功能的关系具有重要的意义。虽然AOH基因在酿酒酵母中成功获得表达,但其表达水平含量低,难以获得足够量的纯化C11α-羟化酶用于结晶及蛋白结构分析。通过TMHMM Server软件预测AOH基因的N端具有33氨基酸的跨膜区。为了增加C11α-羟化酶可溶性表达,对跨膜区域的33个氨基酸分别进行了 20、25和33氨基酸的截短删除。截短体AOH-33、AOH-25、AOH-20重组酵母都能够成功转化甾体底物,但对甾体底物16α,17α-环氧黄体酮的转化水平都低于AOH基因非截短体重组酵母。为了增加赭曲甾体C11α-拜化酶的表达水平,我们进一步尝试大肠杆菌表达体系。分别构建了完整AOH基因和截短体AOH-20、AOH-25、AOH-33相应的大肠杆菌表达载体,并获得了相应的重组菌。由于羟化酶必须和还原酶共同作用才能转化甾体底物,同时构建了赭曲AOR基因表达载体并获得了相应的大肠杆菌重组菌。以甾体底物16α,17a-环氧黄体酮来检测重组菌的甾体C11α羟化活性,未能观察到甾体C11α羟化产物积累。通过SDS-PAGE检测分析显示赭曲AOR还原酶以包涵体形式表达,而AOH及截短体AOH-33、AOH-25、AOH-20在大肠杆菌中未能实现表达。
[Abstract]:Aspergillus ochratus is used in industry to catalyze 16 alpha, 17 alpha epoxy progesterone C11 alpha hydroxylation, synthesis of important glucocorticoid intermediate C11 alpha hydroxy -16 a, 17 alpha epoxy progesterone. The ochratochroma hydroxylase involved in steroid C11 alpha hydroxylation is cytochrome P450 enzyme. This topic first expressed C1 1 alpha hydroxylase in Saccharomyces cerevisiae. Gene AOH, the results showed that AOH recombinant yeast cells could transform 16 alpha, 17 alpha epoxy progesterone forming product C1 1 alpha -16 alpha and 17 alpha epoxy progesterone. It shows that the AOH gene has realized functional expression in yeast cells. The relationship between the structure and function of ochre steroid C11 alpha hydroxylase is of great significance. However, AOH gene was successfully expressed in Saccharomyces cerevisiae, but its expression level was low. It was difficult to obtain enough purified C11 alpha hydroxylase for crystallization and protein structure analysis. The N terminal of the AOH gene was predicted to have 33 amino acid transmembrane regions by TMHMM Server software. In order to increase the soluble expression of C11 alpha hydroxylase, 33 of the transmembrane regions were used. The amino acids were deletions of 20,25 and 33 amino acids respectively. The truncated AOH-33, AOH-25, and AOH-20 recombinant yeast were able to successfully convert the steroid substrates, but the conversion level of the steroid substrate 16 alpha, 17 alpha epoxy progesterone was lower than the AOH gene non truncated recombinant yeast. In order to increase the expression level of ochreosteroid C11 alpha wordase, we The expression vector of Escherichia coli AOH and truncated AOH-20, AOH-25, and AOH-33 was constructed, and the corresponding recombinant bacteria were obtained. The hydroxylase must be combined with the reductase to convert the steroid substrate, and the ochre AOR gene expression vector was constructed and the corresponding vector was constructed. The recombinant bacteria of Escherichia coli. The steroid C11 alpha hydroxylation activity of the recombinant bacteria was detected by the steroid substrate 16 alpha, 17a- epoxy progesterone. The accumulation of the hydroxylation products of steroid C11 alpha was not observed. The SDS-PAGE detection analysis showed that ochre AOR reductase was expressed in the form of inclusion body, and AOH and AOH-33, AOH-25 and AOH-20 were not realized in Escherichia coli. Expression.

【学位授予单位】：天津科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78;Q55

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