Electroenzymatic Activity of Cytochrome P-450cam and Application to the Organic Synthesis

在本项目中，我们构建了一个生物反应器，将还原电子提供给固定化细胞色素P-450cam和/或细胞色素P-450cam，并研究了酶电极的性质。用聚丙烯酸和合适的连接物将腐胺还蛋白、细胞色素P-450cam和细胞色素P-450cam杂化蛋白固定在碳电极上，用循环伏安法分析了这些固定化蛋白的电化学性质。在细胞色素P-450cam存在下，固载腐还蛋白电极发生了可逆的电子转移，氧化还原电位发生了移动。这些结果表明，腐达还蛋白与细胞色素P-450cam之间存在相互作用。而在细胞色素P-450cam电极上，只在第一次扫描时才观察到还原峰。细胞色素P-450CaM固定化电极的电酶活性较低，并检测到过氧化氢的生成。我们构建了恶臭还蛋白-细胞色素P-450cam的融合基因，并在大肠杆菌中表达，纯化了杂合蛋白。杂化蛋白固定化酶电极的循环伏安曲线与细胞色素P-450cam电极相似，但在樟脑存在下略有可逆。杂化蛋白电极的电酶活性是细胞色素P-450cam电极的10倍以上。杂化蛋白电极的过氧化氢生成量很少或没有。这些结果表明，杂化蛋白中的恶臭还蛋白部分可调节细胞色素P-450cam的活性，并抑制自氧化途径细胞色素P-450cam的反应周期。本研究证明了电化学驱动细胞色素P-450用于有机化合物合成的可行性

In this project, we constructed a bioreactor where the reduction electron is supplied to immobilized cytochrome P-450cam and/or putidaredoxin and investigated the character of the enzyme electrode.Putidaredoxin, cytochrome P-450cam and putidaredoxin-cytochrome P-450cam hybrid protein were immobilized on carbon electrode using polyacrylic acid and appropriate linkers, then electrochemical characters of these immobilized proteins were analyzed by cyclic voltammetry. Reversible electron transfer was observed on putidaredoxin immobilized electrode and redox potential of immobilized putidaredoxin was shifted at presence of cytochrome P-450cam in bulk solution. These results indicated interaction between putidaredoxin and cytochrome P-450cam. However, in cytochrome P-450cam electrode reduction peak was only observed on first scan. The elctroenzymatic activity of cytochrome P-450cam immobilized electrode was low and formation hydrogen peroxide was determined. We constructed fusing CDNA of putidaredoxin-cytochrome P-450cam and expressed hybrid protein in Escherichia coli, purified. Cyclic voltammgram of hybrid protein immobilized enzyme electrode was similar to cytochrome P-450cam electrode, but slightly reversible at presence of camphor. Electroenzymatic activity of hybrid protein electrpde was more than 10 times higher than cytochrome P-450cam electrode. Formation of hydrogen peroxide of hybrid protein electrode was little or no. These results suggested that putidaredoxin part of hybrid protein modulate cychrome P-450cam activity and suppressed autooxidation route cytochrome P-450cam reaction cycle.This present research demonstrated the feasibility of electrochemically driven cytochrome P-450 for syntheses of organic compounds