Enzymatic Activity of Lipase−Nanoparticle Conjugates and the Digestion of Lipid Liquid Crystalline Assemblies

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Abstract

Variants of lipase were attached to gold nanoparticles (NPs) and their enzymatic activity was studied. The two bioengineered lipase variants have been prepared with biotin groups attached to different residues on the protein outer surface. The biotinylation was evidenced by denaturing polyacrylamide gel electrophoresis and quantified by the ([2-(4'-hydroxyazobenzene)]benzoic acid spectrophotometric test. NPs of 14 +/- 1 nm diameter coated with thiolated-polyethylene glycol ligands containing controlled proportions of biotin moieties have been prepared and characterized by transmission electron microscopy, UV-vis spectroscopy, small angle neutron scattering, and elemental analysis. These biotin-functionalized NPs were conjugated to lipase using streptavidin as a linker molecule. Enzyme activity assays on the lipase-nanoparticle conjugates show that the lipase loading and activity of the NPs can be controlled by varying the percentage of biotin groups in the particle protecting coat. The lipase-NP conjugates prepared using one variant display higher activity than those prepared using the other variant, demonstrating orientation-dependent enzyme activity. Cryogenic transmission electron microscopy was used to visualize the enzymatic activity of lipase-NP on well-defined lipid substrates. It was found that lipase-coated NPs are able to digest the substrates in a different manner in comparison to the free lipase.

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