Physics And Chemistry Basis Of Biotechnology - De Cuyper - tiera.ru

Sheila J. Sadeghi et al
modulating the desired properties of the biosensor using protein engineering on the one
hand, together with novel interfacial technologies on the other.
2. Engineering artificial redox chains
For potential applications of cytochromes P450 in biosensors it is more desirable to
replace the biological electron delivery and transport system by artificial ones like
electrochemical [99] or photochemical systems [111-112]. Both methods have been
applied to cytochrome P450 since the early years of P450 research. Several laboratories
have used various methods to reduce cytochromes P450 electrochemically [ 113-117].
Although some electrochemical aspects of P450s were reported more than 20 years ago
[118-119], the direct, non-promoted electrochemistry of P450 is rather difficult to
obtain with unmodified electrodes. The enzyme does not interact with the electrode and
is denatured.
The first direct electrochemistry in solution at the edge-plane graphite electrode was
reported by Hill’s group [113]. Rustling’s group has found that P450cam incorporated
in lipid or polyelectrolyte film displayed the well-defined redox behaviour from its
haem Fe(II/III) [ 114]. More recently, Hill’s group [115] demonstrated cyclic
voltammograms on an edge-plane graphite electrode for various P450cam mutants.
The P450 enzyme of interest in the work carried out in this laboratory is P450 BM3
whose characteristics already covered in the introduction make it very interesting for
biotechnological applications. However, P450 BM3 does not react with electrodes
mainly due to its buried haem. The strategy adopted to tackle this problem makes use of
an engineered, artificial redox chain, where electrons are conveyed to the catalytic unit
via a protein known to interact with the electrode surfaces. This strategy plans to
exploit the knowledge of biological ET for biotechnological purposes. In this strategy, a
redox protein with well-characterised electrochemistry, flavodoxin, is used as a module
to transfer electrons to the P450 unit (Figure 4).
R-H+O 2+2H R- OH + H 2O
Figure 4. Schematic representation of the proposed artificial redox chain assembly.
In order to establish the functionality of the chosen building blocks to be used for the
covalent assembly of the artificial redox chains, the ET between the separate proteins
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