本文關(guān)鍵詞： Amycolatopsissp.CGMCC1149 洛伐他汀 羥基化酶 細(xì)胞色素 P450 基因克隆及表達(dá) 體外催化　出處：《江南大學(xué)》2013年碩士論文　論文類型：學(xué)位論文

【摘要】：無(wú)錫他汀作為本實(shí)驗(yàn)室擁有自主知識(shí)產(chǎn)權(quán)的一種新型羥甲基戊二酰輔酶A還原酶抑制劑，由洛伐他汀經(jīng)擬無(wú)枝酸菌Amycolatopsis sp. CGMCC1149轉(zhuǎn)化而來(lái)，是一種潛在的高效降血脂藥物，其中羥基化酶是催化洛伐他汀羥基化的關(guān)鍵限速酶。本研究運(yùn)用簡(jiǎn)并PCR和SEFAPCR從Amycolatopsis sp. CGMCC1149基因組中克隆獲得羥基化酶全新基因并實(shí)現(xiàn)其在大腸桿菌中表達(dá)，同時(shí)初步建立了洛伐他汀體外催化系統(tǒng)，該系統(tǒng)的建立為進(jìn)一步實(shí)現(xiàn)無(wú)錫他汀大規(guī)模制備奠定基礎(chǔ)。 前期研究運(yùn)用簡(jiǎn)并PCR已獲得羥基化酶基因保守區(qū)片段，在此基礎(chǔ)上，，結(jié)合SEFAPCR克隆獲得羥基化酶基因全序列，共計(jì)1209bp，可編碼403個(gè)氨基酸的蛋白，其分子量為44.8kDa。生物信息學(xué)分析顯示：該基因?yàn)橐粋�(gè)新的羥基化酶基因，屬于細(xì)胞色素P450基因超家族CYP105家族，其中與Amycolatopsis azurea的P450Um-1親緣關(guān)系最近；此酶包含氧結(jié)合區(qū)、離子對(duì)結(jié)合區(qū)及血紅素結(jié)合區(qū)等細(xì)胞色素P450典型功能區(qū)。同時(shí)，利用蛋白同源建模技術(shù)得到羥基化酶的3D模型，該模型包含12個(gè)α螺旋和4個(gè)β折疊，大致分為α螺旋集中區(qū)和β折疊集中區(qū)，其中αFG與BC loop組成底物進(jìn)入通道。另外，該基因密碼子使用呈現(xiàn)一定偏好性，密碼子第三位堿基G、C使用頻率比較高。 為建立洛伐他汀體外催化系統(tǒng)，構(gòu)建了一株羥基化酶重組菌E. coli DH5α(pEtac-p450lov)。該菌質(zhì)粒穩(wěn)定性良好，傳代60次后，質(zhì)粒穩(wěn)定性為92%。CO示差光譜顯示在450nm附近有特征吸收峰，表明羥基化酶正確表達(dá)，并以活性形式存在。同時(shí)對(duì)重組菌E. coli DH5α (pEtac-p450lov)的表達(dá)條件進(jìn)行優(yōu)化，其最適條件為：當(dāng)菌體培養(yǎng)至OD600為0.6，加入0.1mM IPTG進(jìn)行誘導(dǎo)，20°C繼續(xù)培養(yǎng)8h時(shí)羥基化酶表達(dá)量最高，培養(yǎng)基中添加0.5mg/L FeCl2可增加羥基化酶表達(dá)量。 為實(shí)現(xiàn)洛伐他汀的體外催化，分別考查了利用3種不同來(lái)源電子傳遞系統(tǒng)建立的洛伐他汀體外催化系統(tǒng)。結(jié)果顯示，以E. coli電子傳遞系統(tǒng)建立的體外催化系統(tǒng)無(wú)法催化洛伐他汀羥基化；以Amycolatopsis sp. CGMCC1149電子傳遞系統(tǒng)建立的系統(tǒng)可以催化洛伐他汀羥基化，但其催化效率較低；而以鐵氧還蛋白及鐵氧還蛋白還原酶為電子傳遞系統(tǒng)所建立的系統(tǒng)可以實(shí)現(xiàn)洛伐他汀體外催化且催化效率最高。因此，選擇以鐵氧還蛋白及鐵氧還蛋白還原酶建立的體外催化系統(tǒng)作為進(jìn)一步研究對(duì)象，對(duì)其反應(yīng)條件進(jìn)行優(yōu)化，其最適反應(yīng)條件為：溫度30°C，最適pH7.5，反應(yīng)時(shí)間6h，NADH600μM，底物濃度30μM。
[Abstract]:Wuxi statins as a new type of hydroxymethyl glutaryl coA reductase inhibitor has its own intellectual property rights. Lovastatin was transformed by Amycolatopsis sp. CGMCC1149, which is a potential drug for reducing blood lipids. Hydroxylase is the key rate-limiting enzyme to catalyze the hydroxylation of lovastatin. In this study, degenerate PCR and SEFAPCR were used from Amycolatopsis sp. A novel hydroxylase gene was cloned from CGMCC1149 genome and expressed in Escherichia coli. At the same time, the in vitro catalytic system of lovastatin was established, which laid a foundation for the further large-scale preparation of Wuxi statins. In previous studies, degenerate PCR had been used to obtain the conserved region of hydroxylase gene. On this basis, the entire sequence of hydroxylase gene was obtained by SEFAPCR cloning, a total of 1209bp. A protein encoding 403 amino acids with a molecular weight of 44.8 kDa. bioinformatics analysis showed that the gene was a new hydroxylase gene. It belongs to the CYP105 family of cytochrome P450 gene superfamily, and has the closest relationship with P450 Um-1 of Amycolatopsis azurea. The enzyme contains oxygen binding region, ion pair binding region, heme binding region and other typical functional regions of cytochrome P450. At the same time, the 3D model of hydroxylase was obtained by using protein homology modeling technology. The model consists of 12 偽 -helix and 4 尾 -folds, which can be divided into 偽 -helix concentrated region and 尾 -fold concentrated region, in which 偽 FG and BC loop form substrates into the channel. The codon usage of the gene showed a certain preference, and the frequency of use of the third base of the codon was high. In order to establish the in vitro catalytic system of Lovastatin, a recombinant strain of E. coli DH5 偽 pEtac-p450 lovastatin was constructed. The plasmid was stable. After 60 passages, the stability of the plasmid was 92.CO differential spectrum showed that there was a characteristic absorption peak near 450 nm, which indicated that the hydroxylase was correctly expressed. At the same time, the expression conditions of E. coli DH5 偽 pEtac-p450 lovlovv were optimized. The optimum conditions were as follows: the hydroxylase expression was the highest when the cell was cultured to OD600 0.6 and 0.1 mm IPTG was added to induce 20 擄C culture for 8 h. The addition of 0.5 mg / L FeCl2 to the medium increased the expression of hydroxylase. In order to realize the in vitro catalysis of lovastatin, the in vitro catalytic system of lovastatin was investigated using three different electron transfer systems. The in vitro catalytic system based on E. coli electron transport system could not catalyze the hydroxylation of lovastatin. The system based on Amycolatopsis sp. CGMCC1149 electron transport system can catalyze the hydroxylation of lovastatin, but its catalytic efficiency is low. The system with ferredoxin and ferredoxin reductase as electron transport system can achieve the highest catalytic efficiency of lovastatin in vitro. The in vitro catalytic system of ferredoxin and ferredoxin reductase was selected as the further research object, and the reaction conditions were optimized. The optimum reaction conditions were as follows: temperature 30 擄C. The optimum pH was 7.5, the reaction time was 6 h, the NADH was 600 渭 m, the substrate concentration was 30 渭 M.
【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：TQ460.1

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