Layer-by-Layer Assembly of Nanosized Membrane Fractions for the Assessment of Cytochrome P450 Xenobiotic Metabolism

Abstract

Herein, we report the use of sequential layer-by-layer (LbL) assembly to design nanostructured films made of recombinant bacterial membrane fractions (MF), which overexpress cytochrome P450 (CYP) and cytochrome P450 reductase. The ability to incorporate MF in LbL multilayered films is demonstrated by an in situ quartz crystal microbalance with dissipation monitoring using poly- l -lysine or poly- l -ornithine as a polycation. Results show that MF preserve a remarkable CYP1A2 catalytic property in the adsorbed phase. Moreover, atomic force microscopy images reveal that MF mostly adopt a flattened conformation in the adsorbed phase with an extensive tendency to aggregate within the multilayered films, which is more pronounced when increasing the number of bilayers. Interestingly, this behavior seems to enhance the ability of embedded MF to remain active after repeated uses. The proposed strategy constitutes a practical alternative for the immobilization of active CYP enzymes. Besides their fundamental interest, MF-based multilayers are useful nano-objects for the creation of new biomimetic reactors for the assessment of xenobiotic metabolism.

Enzyme-assisted mineralization of calcium phosphate: exploring confinement for the design of highly crystalline nano-objects

“…6 The use of track-etched polymer membranes 7 is, indeed, an efficient way to produce monodisperse objects with a high aspect ratio: their diameter is defined by the nanopore size (from 30 nm up to a few µm) and their length by the template thickness (from 5 to 25 µm). The LbL technique offers the possibility to incorporate a variety of (macro)molecules of biological interest, as previously demonstrated for globular 8 and fibrillar proteins, 9,10 enzymes, 11,12 biomembrane fractions, 13 virus, 14 etc. (for recent review see ref.…”

Enzymatic Approach in Calcium Phosphate Biomineralization: A Contribution to Reconcile the Physicochemical with the Physiological View

“…Broaching the biomineralization issue following an enzymatic approach is a powerful way to provide a better understanding of complex biological mechanisms. The enzymatic approach has been explored in numerous fields, for instance, to unravel the microbial activity of biofilms [ 139 , 140 ], to investigate the mechanism of electron/proton transport involved in the respiratory chain [ 141 , 142 , 143 ], to assess the xenobiotic metabolism [ 144 ], etc.…”

Design of Switchable Enzyme Carriers Based on Stimuli-Responsive Porous Polymer Membranes for Bioapplications

“…Physical stimuli possess several intrinsic advantages over chemical stimili: noncontact, tunable strength, and temporal and spatial resolution, the SR-PPMs undergo either a reversible or irreversible modification because of bonding, hydrophobic, or hydrophilic interactions, leading to changes in structure or self-assembly at large-length scales. Ideal SR-PPMs can be fabricated via functionalization of the film surface or pores wall (Figure ) and LBL-assembling methods to improve the surface hydrophilicity and to tune the surface charge, which is beneficial for enzyme immobilization. − …”