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

Abstract

A new organized biomimetic nanostructure embedding a monoclonal antibody in a lipidic matrix has been designed to sequester a hydrophilic enzyme in an oriented position and allowed to preserve the enzyme activity over a few months. The nanostructure was constituted of a glycolipid and a noninhibitory monoclonal IgG directed against the soluble form of acetylcholinesterase (AChE). A mixed monolayer (IgG-glycolipid) was obtained by spreading mixed IgG-glycolipid vesicles at the air/buffer interface. Several measurements (π-A isotherms, surface potential measurements, and compression-decompression cycles) allowed us to demonstrate the presence of IgG in the monolayer, as well as a reorientation of IgG molecules during the compression. After transfer on solid supports by the Langmuir-Blodgett technique, the presence of IgG in the mixed monolayer was characterized by ATR FTIR spectroscopy. Linking of the AChE on the IgG-glycolipid matrix was realized by immunoaffinity, and the enzyme was shown to retain its activity. The opportunity to detect a strong enzymatic activity, even after transfer at high surface pressures, suggested a preferential orientation of the antibody, favorable to retain the enzyme active at the surface of the nanostructure. The homogeneity of the transferred monolayer before and after immunoassociation, observed by Nomarski microscopy, did not display any structural modification. The enzyme kinetics was typical of the biocatalytic behavior of an immobilized enzyme, with a decrease of reaction rates due to the lower accessibility to the substrate at higher enzyme content. With the advantages of stability and favorable orientation of IgG, this new active matrix induces, in turn, a favorable orientation of the enzyme bound by immunoaffinity. The typical enzymatic behavior of the ternary nanostructure (glycolipid-IgG-AChE) demonstrates the usefulness of such a functional molecular assembly for biocatalysis study in a biomimetic situation.

Kinetics Study of Bungarus fasciatus Venom Acetylcholinesterase Immobilised on a Langmuir–Blodgett Proteo‐Glycolipidic Bilayer

“…[18] In this work, modelling of this molecular assembly has confirmed the molecular arrangement proposed on the basis of experimental data. [18] Indeed, the functional orientation of AChE-IgG immune complexes in the bilayer structure in silico is plausible with respect to the steric hindrance induced by the immobilization of several complexes. The main characteristic of this molecular association is to maintain a hydrophilic enzyme close to the lipidic membrane in an orientated position.…”

“…[18] Mechanical dispersion was used to prepare glycolipid and proteo-glycolipidic vesicles at room temperature by the previously described procedure. [40] Briefly, glycolipid vesicles and IgG-glycolipid vesicles were prepared by dispersion of a dry glycolipid film (2.5 mg) either in phosphate buffer (10 mm, pH 7.4, 250 mL), or in the purified antibody solution (3.5-4 mgmL À1 , 250 mL), respectively.…”

“…This microenvironment effect induced an increase in the substrate concentration (S) at which the rate of the heterogeneous enzymatic reaction was half the saturation rate. This particular substrate concentration, in some cases misleadingly termed apparent K M , is named S 0.5 and has largely been interpreted by Engasser and Horvath, [14] or in our group, [15][16][17][18] in terms of substrate diffusion from the bulk solution towards the catalytic sites. One proposed method to reduce the diffusional effects on the enzyme activities measured in the bulk is through more efficient stirring of the reaction medium.…”

“…[18] By inserting a noninhibitor monoclonal antibody (IgG) directed against the soluble acetylcholinesterase monomer (AChE), this structure enables the enzyme to be bound in a functional position at the surface of the lipidic matrix. Previous results have shown that the enzyme stability allows the direct measurement of the AChE activity over a long period of time.…”

“…Previous results have shown that the enzyme stability allows the direct measurement of the AChE activity over a long period of time. [18] In this paper, modelling of the ternary biomimetic molecular assembly (AChE-IgG-glycolipid) has been achieved for the first time, providing insights into the molecular orientation of the AChE-IgG immune complex in the glycolipid bilayer. The stability of the ternary nanostructure as a function of the hydrocarbon chain length of the neoglycolipid molecule was then investigated in order to define experimental conditions giving access to the kinetic properties of the immobilised enzyme.…”