新型羟基化酶基因的克隆及其洛伐他汀转化的应用

Cloning and application of a novel hydroxylase in lovastatin conversion

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。

Wuxistatin, a novel and potent statin, is converted from lovastatin by Amycolatopsis sp. CGMCC1149. In the bioconversion, lovastatin is firstly hydroxylated by a hydroxylase. To obtain the critical hydroxylase, a novel hydroxylase gene was isolated t^om Amycolatopsis sp. CGMCC1149 by Degenerate PCR and Self-Formed Adaptor PCR and expressed in Escherichia coli. BLAST sequence analysis revealed that the gene belonged to cytochrome P450 gene superfamily and could encode a 403-amino-acid protein with a molecular weight of 44.8 kDa. The secondary structure prediction result showed that this protein contained many typical functional regions of P450, such as oxygen binding site, ion-pair region and heme binding region. Meanwhile, a catalytic function verification system was constructed by NADH, ferredoxin and ferredoxin reductase which could catalyze lovastatin hydroxylation into the target product. These would be helpful for further studies in large-scale production of wuxistatin.