(362d) Biocatalytic Coatings of Cytochrome P450-PNIPAM Diblock Copolymers

Engineering proteins into functional catalysts enables their application as biosensors and green catalysts. The biocatalyst must maintain mechanical integrity, protein stability and longevity, and access to the protein active site in order to be a viable technology. In addition, a high protein density is needed to achieve high catalytic activity. The self-assembly of proteins in solid-state materials has the ability to achieve each of these requirements. The modification of proteins with synthetic polymers, creating a protein-based block copolymer, can be used to direct both the solution and solid state self-assembly of proteins. As a demonstration of the utility of this technique, cytochrome P450 BM3 was selected as an attractive target for a functional immobilized enzyme. The bacterial P450 contains both oxidase and reductase domains, has been extensively engineered to accept non-native substrates and catalyze novel chemical transformations, and has been evolved to have improved thermostability. Matching these catalytic improvements to improvements in enzyme immobilization technology could enable the use of P450s as biocatalysts. Functionalization of the enzyme with the synthetic polymer poly(N-isopropylacrylamide) creates a bioconjugate that can self-assemble to form functional nanostructured films. The self-assembly of the bioconjugate in bulk and concentrated solutions was characterized by small-angle x-ray scattering (SAXS) and depolarized light scattering (DPLS). The synthetic conjugate also enables the enzyme to be flow coated into a thin film that is readily immobilized by a simple chemical crosslinking step. The activity of the self-assembled P450 thin films was assayed for a variety of substrates.