First steps towards a Zn/Co(III)sep-driven P450 BM3 reactor

Area of Science:

• Biocatalysis
• Enzyme Engineering
• Chemical Synthesis

Background:

• Cytochrome P450s are valuable hydroxylation catalysts, but their cell-free applications are limited by the high cost of NAD(P)H cofactor regeneration.
• Mediator systems, such as Zn/Co(III)sep, offer a cost-effective alternative cofactor system for P450-catalyzed reactions.

Purpose of the Study:

• To engineer and immobilize cytochrome P450 BM3 mutants for enhanced electron transfer with mediator systems.
• To develop stable and efficient biocatalytic systems for cell-free hydroxylation reactions.
• To explore the synthetic potential of engineered P450 BM3 in continuous flow bioreactors.

Main Methods:

• Engineering of P450 BM3 mutants (M7 and M9) with improved electron transfer kinetics.
• Immobilization of P450 BM3 M7 on DEAE-650S and entrapment with k-carrageenan, zinc dust, and catalase.
• Operational stability assessment of immobilized P450 BM3 M7 over 10 batch cycles.
• Development of a plug flow reactor (PFR) for continuous conversion using P450 BM3 M9.

Main Results:

• Immobilized P450 BM3 M7 retained 76% activity and >80% conversion over 10 batch cycles, demonstrating high operational stability.
• P450 BM3 M9 exhibited a 3-fold higher activity for 3-phenoxytoluene compared to P450 BM3 M7.
• The PFR system using P450 BM3 M9 achieved total turnover numbers (TTNs) over 2,000 during 5 days of continuous operation.

Conclusions:

• Engineered and immobilized P450 BM3 variants, coupled with a mediator system, provide a stable and efficient platform for cell-free hydroxylation.
• The developed continuous flow system demonstrates significant potential for industrial applications in biocatalysis.
• This approach overcomes the limitations of costly cofactor regeneration, enabling more sustainable chemical synthesis.