Expanding an Efficient, Electrically Driven and CNT‐Tagged P450 System into the Third Dimension: A Nanowired CNT‐Containing and Enzyme‐Stabilising 3 D Sol–Gel Electrode

Abstract

Although electrochemically catalysed P450 reactions have been described, their efficiency and applicability remained limited. This is mostly due to low enzyme activity, laborious protein immobilisation and the small electrode surface. We established a novel protein immobilisation method for a determined orientation and electrical wiring of the enzyme without post-expression modification. By genetic introduction of an anchor-peptide our method is applicable for screening medium to large mutant libraries and detection by an electrode system. The system was expanded by using wired carbon nanotubes within a sol-gel matrix to create a three dimensional electrode.

Enzymatic Hydroxylations of sp3-Carbons

“…Another approach is the coexpression of glucose dehydrogenase within a whole cell setup to facilitate NADPH-regeneration, resulting in an approximately 22-fold increase in product concentration . Switching the cofactor from NADPH to NADH or other cofactors, such as N -benzyl-1,4-dihydronicotinamide, might be beneficial, if applicable, as they are more stable and can reduce process costs. − The immobilization of P450s on electrodes was performed to investigate and further improve the electrochemical performance by fine-tuning their catalytic parameters and substrate recognition leading to possible applications as amperometric sensors. , Another possibility, which has been investigated in detail, is the replacement of NADPH by a light-generated source of electrons. This substitution is possible as the ferredoxin-mediated electron transfer in mitochondrial and some bacterial P450 systems is similar to the electron carriers in the photosynthetic chain.…”

An Overview of Cytochrome P450 Immobilization Strategies for Drug Metabolism Studies, Biosensing, and Biocatalytic Applications: Challenges and Opportunities

“…154 CNTs have been used to orient P450 BM3 on an electrode by genetic fusion of a high-affinity CNT-binding peptide. 131 To increase the density of immobilized P450s, a 3D immobilization platform based on a sol−gel was built on the electrode, containing CNTs wired by the conductive polymer poly (3,4-ethylenedioxythiophene) designed to increase the density of immobilized P450s. After 20 h, the sol−gel immobilized enzyme showed the same activity as freshly entrapped enzyme.…”

“…Interestingly, the approach was applied to screen a library of P450 BM3 variants for the hydroxylation of aniline to aminophenol; compatibility with a microtiter plate-format promises broader application of the platform. 131 Immobilization with His-tags is pH-dependent, limiting their application. Z basic2 is an engineered, arginine-rich tag with a high isoelectric point (pI > 9), designed to circumvent this limitation.…”

Prospects for Applying Synthetic Biology to Toxicology: Future Opportunities and Current Limitations for the Repurposing of Cytochrome P450 Systems

“…The electrochemical properties of cytochrome P450 enzymes provide a means to monitor the P450 catalytic cycle electronically, and several P450 biosensor systems have been developed along these lines. − Much of the research in this area , has been motivated by the desire to deliver electrons to P450s using electrodes rather than consuming expensive cofactors during catalysis; where successful, catalysis can be achieved either (1) by the generation of hydrogen peroxide by reduction of Fe­(II)-O 2 at the electrode and its subsequent use to generate the high valent, active, oxygenating species of P450 (compound I) via the peroxide shunt (which can occur in the absence of any redox partner) or (2) in a way that more closely mimics the native system, i.e., via reduction of the P450 by CPR reduced at the electrode. , It is unclear what the working lifetime of such systems would be if employed for biocatalysis, especially in light of the continual production of hydrogen peroxide at the electrode surface via electrolysis of oxygen. Therefore, it would be advantageous to engineer forms of the enzyme that are resistant to oxidative damage and also more thermostable for further development of such systems.…”