Influence of Lipidic Matrix and Structural Lipidic Reorganization on Choline Oxidase Activity Retained in LB Films

Abstract

This work focuses on the influence of the nature of the lipidic matrix and of the hydrophobic or hydrophilic lipidic environment on choline oxidase activity retained in Langmuir−Blodgett (LB) films prepared with behenic acid or with mixed dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidic acid (DPPA). The enzyme activity was detected through an electrochemical sensor whose platinum electrode was coated with the protein−lipid LB films. It was shown that the hydrophilic environment provided by the phospholipidic LB films was directly responsible for an inactivation of choline oxidase likely due to a competition with the choline moiety of DPPC. The environment provided by the carboxylic groups of the fatty acid multilayers allowed retention of the higest activity. The absence of stability of choline oxidase activity in behenic acid LB films was explained by repulsive electrostatic interactions between the protein and the lipidic molecules, which appeared during the immersion of the LB films in the alkaline buffer required to detect the enzyme activity in optimal conditions. These unfavorable interactions, previously shown to be responsible for a lipidic structural reorganization of choline oxidase−behenic acid LB films, seem to be here responsible for a likely unfolding, yielding protein denaturation. Because it controls the electrostatic interactions between the protein and the lipidic molecules, which may be very unfavorable even in the optimal conditions of the enzyme activity, pH acts as a key parameter of the expression of the enzyme activity in the LB films.

New Functional Proteo-glycolipidic Molecular Assembly for Biocatalysis Analysis of an Immobilized Enzyme in a Biomimetic Nanostructure

“…No enzyme desorption was observed when the plate was removed from the enzyme substrate medium. This strong retention can be attributed to the specificity of IgG−AChE recognition, to the strong proteolipidic interactions between the IgG and the glycolipid, and to the absence of charge in the glycolipid film, thus avoiding reorganization , or denaturation due to charge repulsions, as previously reported.…”

Surface density as a significant parameter for the enzymatic activity of two forms of alkaline phosphatase immobilized on phospholipid Langmuir–Blodgett films

“…The solid support and the enzyme immobilization should avoid enzyme denaturation and must also provide an orientation and enzymatic surface density that maximize the catalytic activity. Furthermore, the reproducibility of the support preparation and the homogeneity of the interface are also important aspects for a detailed understanding of the immobilized enzyme behavior [15,16].…”

Layered Biosensor Construction

“…In order the aim to develop models mimicking biomembranes usable for applications in the biosensor field, studies of biological activities of membraneous proteins after incorporation in a phospholipidic bilayer were widely investigated [29,30]. Well known is the sensing system built by the incorporation of glutamate dehydrogenase or choline oxidase into fatty acid LB films through an adsorption or an inclusion process which consists of sandwiching the protein molecules between two LB layers [31]. The molecules of glutamate dehydrogenase adsorbed on the surface of behenic acid LB films work as a protective screen against the rearrangement of the multilayers induced by diffusion of the alkaline buffer inside the structure; on the contrary, the choline oxidase molecules operate as an accelerating factor of the structural lipidic reorganization induced in the same conditions.…”