Supramolecular protein assembly supports immobilization of a cytochrome P450 monooxygenase system as water-insoluble gel

Scientific Reports
Cheau Yuaan TanTeruyuki Nagamune

Abstract

Diverse applications of the versatile bacterial cytochrome P450 enzymes (P450s) are hampered by their requirement for the auxiliary proteins, ferredoxin reductases and ferredoxins, that transfer electrons to P450s. Notably, this limits the use of P450s as immobilized enzymes for industrial purposes. Herein, we demonstrate the immobilization of a bacterial P450 and its redox protein partners by supramolecular complex formation using a self-assembled heterotrimeric protein. Employment of homodimeric phosphite dehydrogenase (PTDH) for cross-linking "proliferating cell nuclear antigen-utilized protein complex of P450 and its two electron transfer-related proteins" (PUPPET) yielded a gelling PUPPET-PTDH system capable of regenerating NADH for electron supply owing to its phosphite oxidation activity. The protein gel catalyzed monooxygenation in the presence of phosphite and NAD(+). The gel was completely water-insoluble and could be reused. This concept of oligomeric protein-insolubilized enzymes can be widely applied to various multienzymatic reactions such as cascade reactions and coupling reactions.

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Citations

Oct 7, 2015·ACS Applied Materials & Interfaces·Sailendra Mahanta, Subhankar Paul
Jul 16, 2016·Microbial Biotechnology·Claudia Schmidt-Dannert, Fernando Lopez-Gallego
Apr 5, 2017·Organic & Biomolecular Chemistry·M B QuinC Schmidt-Dannert
Oct 27, 2016·Angewandte Chemie·Cheau Yuaan TanTeruyuki Nagamune
Aug 2, 2018·Applied Microbiology and Biotechnology·Sarah Schmidt-DannertClaudia Schmidt-Dannert
Dec 15, 2018·Current Opinion in Chemical Biology·Claudia Schmid-Dannert, Fernando López-Gallego

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Methods Mentioned

BETA
size-exclusion chromatography
size exclusion chromatography
dynamic light scattering

Software Mentioned

GraphPad Prism
ImageJ

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