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无锡他汀由洛伐他汀经拟无枝酸菌Amycolatopsis sp.CGMCC1149羟基化而来。为获得该转化过程中的关键酶——羟基化酶,运用简并PCR和SEFA PCR技术,从Amycolatopsis sp.CGMCC1149中克隆获得长度为1 212 bp的新型羟基化酶基因,并实现其在大肠杆菌中表达。BLAST序列分析表明该基因属于细胞色素P450基因超家族,并可编码一个含403个氨基酸的蛋白,其分子量为44.8 kDa。二级结构预测结果表明:该蛋白包含有氧结合区、离子对结合区和血红素结合区等P450典型功能区。同时文中利用NADH、铁氧还蛋白和铁氧还蛋白还原酶建立了体外酶催化功能验证系统,首次实现无锡他汀转化底物洛伐他汀的体外羟基化。该结果为具有我国独立知识产权的无锡他汀大规模制备奠定了基础。
Wuxi statins by Lovastatin Amycolatopsis sp. CGMCC1149 hydroxylation. In order to obtain the key enzyme in this transformation, hydroxylase, a novel hydroxylase gene with a length of 1 212 bp was cloned from Amycolatopsis sp. CGMCC1149 by degenerate PCR and SEFA PCR, In the expression. BLAST sequence analysis showed that the gene belonged to cytochrome P450 superfamily and encoded a 403 amino acid protein with a molecular weight of 44.8 kDa. The secondary structure prediction results show that the protein contains P450 typical functional regions such as oxygen binding region, ion-binding region and heme binding region. In the meantime, an in vitro enzymatic functional verification system was established by using NADH, ferredoxin and ferredoxin reductase, and the in vitro hydroxylation of lovastatin, a substrate for conversion of Wuxi statins, was realized for the first time. The results laid the foundation for the large-scale preparation of statins in Wuxi with independent intellectual property rights in China.