本科毕业设计（论文）来源于巨大芽孢杆菌ALA2的细胞色素P450-BM-3的蛋白质工程研究.doc

本科毕业设计（论文） 题 目： 来源于巨大芽孢杆菌ALA2的 细胞色素P450 BM-3的蛋白质工程研究 学 院： 专 业： 学 号： 学生姓名： 指导教师： 职 称： 年 月 日  摘 要 细胞色素P450酶系是广泛分布于动物、植物和微生物等不同生物体内的一类代谢酶系。来源于巨大芽孢杆菌的细胞色素3可以快速催化链长大约C12本实验采用来源于巨大芽孢杆菌ALA2的细胞色素P-3基因进行蛋白质工程设计，按此依据进行定点突变和重组表达，并和原始基因在大肠杆菌中表达情况进行对比相比于型细胞色素P-3，F87对吲哚显示了高的活性，由此证明Phe87对反应选择性控制起着关键的作用，是影响该酶表达量和表达活力的主要因素。巨大芽孢杆菌The study on protein engineering of the cytochrome P450 BM-3 from B. megateriummALA2 Abstract Cytochrome P450 is a kind of metabolic enzymes which are widely distributed in animals, plants and microorganisms. Cytochrome P450 BM-3 from B. megateriumm shows high monooxygenase activity for long chain saturated fatty acid(12-18 carbons), especially fatty acid with 15 and 16 carbons are the highest. P450 BM-3 encoding gene (CYP102) has been cloned and expressed in E. coli, the purified P450 expressed from E. coli has the same function, immunochemistry and electrophoresis characteristic with the purified P450 BM-3 from B. megateriumm, and their N-terminal sequences are also consistent. The study usesd the cytochrome P450 BM-3 gene from B. megateriummALA2 for protein engineering design, according to which we conducted site-directed mutagenesis and recombination expression,and compared with the original gene expression in E. coli. F87L shows a litter higher activity towards indole than wild-type P450 BM-3, and the enzyme activity has increased by 6.98%, which proved that Phe87, playing a key role in the control of reaction selectivity, is the major factor influencing the enzyme expression and activity. The study also lay a good foundation to further exploration of cytochrome P450 BM-3 mutant for biocatalysis application. Keywords: Bacillus. Megaterium,P450 BM-3,F87L,indole 目 录 摘 要 I Abstract II 前 言 1 1 文献综述 2 1.1 细胞色素P450 2 1.1.1 细胞色素P450的酶系组成 2 1.1.2 细胞色素P450的结构折叠 2 1.1.3 细胞色素P450的底物结合与识别 3 1.1.4 细胞色素P450的天然底物 3 1.1.5 细胞色素P450的催化反应 4 1.1.6 细胞色素P450基因的简化结构 5 1.2 细胞色