Electrochemical Regeneration of an Enzyme Cosubstrate Peroxidase Activity

A carbon felt modified by electro-deposition of a polypyrrole biotin layer to which biotinylated horseradish peroxidase (HRP) is attached by the molecular recognition process involving the biotin-avidin interaction (described in Chap. 1) has been introduced in a simple reactor where a solution of the enzyme reactants (H2O2 and Fe(CN)6K4) flow across the enzyme-modified carbon felt (Fig. 30). The conducting enzyme support is connected to a potentiometer as a working electrode biased to a potential at which the reduction of Fe(CN)6K3 occurs. The reaction is initiated by the addition of a low concentration of Fe(CN)6K4 to start the enzyme catalytic reaction, and the enzyme turnover is maintained by the electrochemical coupling, as the cosubstrate of the HRP: Fe(CN)6K4 is electrochemically regenerated from the oxidized form Fe(CN)6K3 formed in the reactor. The electrochemical coupling process is shown in Fig. 30.

Fig. 30. Scheme of the working principle of peroxidase (HRP) immobilized on carbon felt covered with a polypyrrole biotin polymer. The HRP is attached to the polymer surface by biotin-avidin molecular recognition linkage. The regeneration of the redox mediator Fe(CN)6- is achieved biasing the carbon felt at -0.2 V versus an Ag/AgCl electrode

Fig. 30. Scheme of the working principle of peroxidase (HRP) immobilized on carbon felt covered with a polypyrrole biotin polymer. The HRP is attached to the polymer surface by biotin-avidin molecular recognition linkage. The regeneration of the redox mediator Fe(CN)6- is achieved biasing the carbon felt at -0.2 V versus an Ag/AgCl electrode

The variation of the Fe(CN)6K3 concentration is followed by absorption spectroscopy at 305 nm. Depending on the initial concentration of the Fe(CN)6K4 cosubstrate, for a given initial H2O2 concentration, the production of oxidized Fe(CN)6- species increases (see Fig. 31), which is an indication of the activity of the immobilized peroxidase. After a given time, the concentration of Fe(CN)6K3 decreases in the reactor as a result of the electrocatalytic coupling effect regenerating the cosubstrate. This effect is also evidenced by the reduction in the concentration ofH2O2 as the enzyme activity is started.

The concentration of Fe(CN)6K3 affects the initial slope of the ferri-cyanide production as well as that of the related H2O2 dismutation, but the slope ratios do not vary exactly in the same proportion. The more probable explanation for this is the diffusion-limited process of access of reactants to the immobilized enzymes sites in the core of the grafted textile.

However, the data shown in Fig. 32 indeed confirm the electrocatalysis coupling, as the amount of H2O2 converted is much higher than that of ferricyanide initially introduced in the reactor. After about 50-60 min, as 1 mole of H2O2 necessitates 1 mole of Fe(CN)6K3 to be converted into products, the initial ferricyanide concentration introduced in the reactor has been approximately regenerated 100 times by the electrical reduction coupling at the carbon felt surface.

Fe(CN)^ absorbante with KO 10J M

Fig. 31. Variation with time of the potassium ferricyanate absorbance for different initial concentrations of Fe(CN)6K4 in 10-3 M H2O2 solution (the carbon felt is electrically biased at -0.2 V versus Ag/AgCl electrode)

Time(min)

Fig. 31. Variation with time of the potassium ferricyanate absorbance for different initial concentrations of Fe(CN)6K4 in 10-3 M H2O2 solution (the carbon felt is electrically biased at -0.2 V versus Ag/AgCl electrode)

Fig. 32. Change in the H2O2 substrate concentration during the HRP reactivity under coupling with electrocatalysis

Electrochemical Production of an Enzyme Substrate

Another illustration of the interest of the electrical connection of the en-zymetextilesupport isthepossibleproduction, forinstance, of thesubstrate of a peroxidase activity. The electrochemical production of H2O2 is used in water treatment processes (Drogui et al. 2001), and it can be used similarly here for the working function of HRP immobilized onto a carbon felt.

The direct reduction of oxygen onto the carbon felt surface was achieved by biasing the substrate at -0.5 V/ECS, where the oxygen reduction occurs according to the classical electrochemical reaction:

The scheme shown in Fig. 33 depicts the working conditions of the enzyme reactor used in association with the electrochemical production of the HRP substrate, the chosen cosubstrate being pyrogallol, and oxygen fed in the reactor by gas bubbling. As in the case of the regeneration of the HRP cosubstrate described in the previous paragraph, the coupling efficiency is evidenced by the data shown in Fig. 32.

One of the products of the enzyme catalytic process, purpurogallin, is clearly formed from the pyrogallol fed into the reactor. The purpurogallin concentration depends on that of the initially introduced pyrogallol, showing that the H2O2 substrate is electrochemically produced in sufficient purpurogallin pyrrogallol

O, purpurogallin pyrrogallol

h20 h2o2

Fig. 33. Scheme of the catalytic reactions on the surface of a modified textile amount from oxygen fed into the reactor, and does not limit the kinetics of the enzyme reaction.

Conclusion

In conclusion, the two examples mentioned in this section demonstrate that the immobilization of enzymes by a molecular recognition process onto textiles composed of electron-conducting fibers can be coupled to electrocatalysis. This coupling is efficient, even if in the described data some kinetic limitations are noticeable, due presumably to limited diffusion processes of species in the vicinity of the enzyme reactive sites in the textile core. However, as these experiments have been achieved in a very simple laboratory enzyme reactor, better optimization of the reactor design should emphasized this point.

Acknowledgements. We are very indebted to N. Bouhaine at the Department of Chemistry, Badji Mokhtar University, BP 12,23000 Annaba, Algérie, and to A. Gherrou at the Faculty of Chemistry, USTHB, 16111 Bab Ezzouar, Algérie for their contributions to the experiments involving trypsin and pepsin grafted onto cotton. The authors also thank Professor Zhi-Kang XU and Dr. Zhen-Mei LIU at the Institute of Polymer Science, Zhejiang University, Hangzhou, 310027 People's Republic of China, for their collaboration in the domain of enzyme modifications of membranes in the frame of the Advanced Research Program (PRA 03-05) between France and China.

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