Study On The Electrochemical Behaviors Of Chloroperoxidase And Its Application

Chloroperoxidase (CPO) from Caldariomyces fumago is a versatile monomeric hemoenzyme has been widely used for catalytic reactions of a broad spectrum of substrates because of its unique hybrid structure. Use of electrochemically generated hydrogen peroxide also shows extended enzyme lifetime compared to direct addition. However, the reaction is costly and impractical for industrial applications because soluble CPO was used for the catalytic reaction and these expensive enzymes could not be reused. The equipment which was used to add hydrogen peroxide also is expensive.In this paper, the CPO was be used to immobilized with the polyions onto the Au-quartz crystal microbalance (QCM) or glassy carbon electrode surface by self assembled technology. Layered polycation-CPO films were grown on Au treated with 3-mercaptopropanesulfonic acids, sodium salt (MPS) ethanol solution, to provide an even negatively charged surface. Then electrode was modified with polydimethyldiallylammonium chloride (PDDA) which was positively charged. And a layer of negatively charged CPO was adsorbed on the positive surface. The frequency shift of Au-QCM demonstrated monolayer formed during each adsorption. The cyclic voltammetric results showed that self assembled layer is a diffusionless behavior. The oxidation-reduction peaks (Epa = 0 mV, Epc=-135 mV) with around 135 mV peak separation, compared with about 60 mV with the film modified on GC electrode.And the biomembrane-like composite films of CPO and didodecyldimethyl- ammonium bromide (DDAB) also used to immobilize the CPO. Direct electron transfer between CPO and electrode has been achieved. As the result, the DDAB-CPO films on the PG electrode was best reversible and stable,ΔEp =60 mV. To investigate the pH dependence, cyclic voltammetry was performed at different pH phosphate buffer solution. An increase in pH of buffer solutions led to a negative shift in both of the reduction and oxidation peak potentials, linear relationship of formal potential versus pH with a slope of 25 mV / pH.The CPO modified electrodes with self-assembled membrane(SAM) and biomembrane-like film DDAB were employed for chlorination of monochlorodimedone (MCD, have UV adsorption at 278 nm) to didochlorodimedone (DCD, no UV adsorption at 278 nm) using a unique electrochemical system which used UV spectrum adsorption to observe the reaction. As the result, CPO immobilized with biomembrane-like film more efficient with the same electrode surface area. The catalyze mechanism of CPO was discussed. In this system hydrogen peroxide was continuously generated at the electrode with keeping at -0.6V. Hydrogen peroxide generated at the electrode oxidate the CPO to active form. Then the active forms convert MCD to DCD with the chlorin ion in the solution.