Amycolatopsis sp.CGMCC1149新型羟基化酶基因的克

发布时间：2018-02-04 05:27

本文关键词： Amycolatopsissp.CGMCC1149 洛伐他汀羟基化酶细胞色素 P450 基因克隆及表达体外催化　出处：《江南大学》2013年硕士论文　论文类型：学位论文

【摘要】：无锡他汀作为本实验室拥有自主知识产权的一种新型羟甲基戊二酰辅酶A还原酶抑制剂，由洛伐他汀经拟无枝酸菌Amycolatopsis sp. CGMCC1149转化而来，是一种潜在的高效降血脂药物，其中羟基化酶是催化洛伐他汀羟基化的关键限速酶。本研究运用简并PCR和SEFAPCR从Amycolatopsis sp. CGMCC1149基因组中克隆获得羟基化酶全新基因并实现其在大肠杆菌中表达，同时初步建立了洛伐他汀体外催化系统，该系统的建立为进一步实现无锡他汀大规模制备奠定基础。前期研究运用简并PCR已获得羟基化酶基因保守区片段，在此基础上，，结合SEFAPCR克隆获得羟基化酶基因全序列，共计1209bp，可编码403个氨基酸的蛋白，其分子量为44.8kDa。生物信息学分析显示：该基因为一个新的羟基化酶基因，属于细胞色素P450基因超家族CYP105家族，其中与Amycolatopsis azurea的P450Um-1亲缘关系最近；此酶包含氧结合区、离子对结合区及血红素结合区等细胞色素P450典型功能区。同时，利用蛋白同源建模技术得到羟基化酶的3D模型，该模型包含12个α螺旋和4个β折叠，大致分为α螺旋集中区和β折叠集中区，其中αFG与BC loop组成底物进入通道。另外，该基因密码子使用呈现一定偏好性，密码子第三位碱基G、C使用频率比较高。为建立洛伐他汀体外催化系统，构建了一株羟基化酶重组菌E. coli DH5α(pEtac-p450lov)。该菌质粒稳定性良好，传代60次后，质粒稳定性为92%。CO示差光谱显示在450nm附近有特征吸收峰，表明羟基化酶正确表达，并以活性形式存在。同时对重组菌E. coli DH5α (pEtac-p450lov)的表达条件进行优化，其最适条件为：当菌体培养至OD600为0.6，加入0.1mM IPTG进行诱导，20°C继续培养8h时羟基化酶表达量最高，培养基中添加0.5mg/L FeCl2可增加羟基化酶表达量。为实现洛伐他汀的体外催化，分别考查了利用3种不同来源电子传递系统建立的洛伐他汀体外催化系统。结果显示，以E. coli电子传递系统建立的体外催化系统无法催化洛伐他汀羟基化；以Amycolatopsis sp. CGMCC1149电子传递系统建立的系统可以催化洛伐他汀羟基化，但其催化效率较低；而以铁氧还蛋白及铁氧还蛋白还原酶为电子传递系统所建立的系统可以实现洛伐他汀体外催化且催化效率最高。因此，选择以铁氧还蛋白及铁氧还蛋白还原酶建立的体外催化系统作为进一步研究对象，对其反应条件进行优化，其最适反应条件为：温度30°C，最适pH7.5，反应时间6h，NADH600μM，底物浓度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 伪 -helix and 4 尾 -folds, which can be divided into 伪 -helix concentrated region and 尾 -fold concentrated region, in which 伪 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 伪 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 伪 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 掳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 掳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.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：TQ460.1

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