本文選題：甾體11α-羥化酶 + 釀酒酵母　； 參考：《天津科技大學》2017年碩士論文

【摘要】：赭曲霉在工業(yè)上用于催化16α,17α-環(huán)氧黃體酮C11α-羥化反應,合成重要糖皮質激素類藥物中間體C11α-羥基-16α,17α-環(huán)氧黃體酮。參與甾體C11α-羥基化反應的赭曲霉羥化酶為細胞色素P450酶。本課題首先在釀酒酵母中異源表達C1 1α-羥化酶基因AOH,結果表明AOH重組酵母細胞能夠轉化16α,17α-環(huán)氧黃體酮形成產(chǎn)物C1 1α-16α,17α-環(huán)氧黃體酮,說明AOH基因在酵母細胞中實現(xiàn)了功能表達。由于赭曲甾體C11α-羥化酶在工業(yè)上的價值,分析該酶的結構與功能的關系具有重要的意義。雖然AOH基因在釀酒酵母中成功獲得表達,但其表達水平含量低,難以獲得足夠量的純化C11α-羥化酶用于結晶及蛋白結構分析。通過TMHMM Server軟件預測AOH基因的N端具有33氨基酸的跨膜區(qū)。為了增加C11α-羥化酶可溶性表達,對跨膜區(qū)域的33個氨基酸分別進行了 20、25和33氨基酸的截短刪除。截短體AOH-33、AOH-25、AOH-20重組酵母都能夠成功轉化甾體底物,但對甾體底物16α,17α-環(huán)氧黃體酮的轉化水平都低于AOH基因非截短體重組酵母。為了增加赭曲甾體C11α-拜化酶的表達水平,我們進一步嘗試大腸桿菌表達體系。分別構建了完整AOH基因和截短體AOH-20、AOH-25、AOH-33相應的大腸桿菌表達載體,并獲得了相應的重組菌。由于羥化酶必須和還原酶共同作用才能轉化甾體底物,同時構建了赭曲AOR基因表達載體并獲得了相應的大腸桿菌重組菌。以甾體底物16α,17a-環(huán)氧黃體酮來檢測重組菌的甾體C11α羥化活性,未能觀察到甾體C11α羥化產(chǎn)物積累。通過SDS-PAGE檢測分析顯示赭曲AOR還原酶以包涵體形式表達,而AOH及截短體AOH-33、AOH-25、AOH-20在大腸桿菌中未能實現(xiàn)表達。
[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.

【學位授予單位】：天津科技大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q78;Q55

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