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

本课题构建了一种为固定化细胞色素P-450 cam和/或putidaredoxin提供还原电子的生物反应器，并研究了该酶电极的性质，采用聚丙烯酸和合适的连接剂将Putidaredoxin、细胞色素P-450 cam和putidaredoxin-细胞色素P-450 cam杂合蛋白固定在碳电极上，用循环伏安法分析了固定化蛋白的电化学性质。在putidaredoxin固定化电极上观察到可逆的电子转移，并且在本体溶液中细胞色素P-450 cam的存在下，固定化putidaredoxin的氧化还原电位发生移动。这些结果表明putidaredoxin和细胞色素P-450 cam之间的相互作用。然而，在细胞色素P-450 cam中，仅在第一次扫描时观察到电极还原峰。细胞色素P-450 cam固定化电极的酶活性较低，并测定了过氧化氢的生成。构建了putidaredoxin-细胞色素P-450 cam融合cDNA，在大肠杆菌中表达并纯化了融合蛋白。杂化蛋白质固定化酶电极的循环伏安曲线与细胞色素P-450 cam电极相似，但在樟脑存在下略有可逆性。杂交蛋白电极的酶活性比细胞色素P-450 cam电极高10倍以上。结果表明，该杂合蛋白的putidaredoxin部分能够调节细胞色素P-450 cam的活性，抑制细胞色素P-450 cam的自氧化途径，从而证明了电化学驱动细胞色素P-450合成有机化合物的可行性

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