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

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.

Preparation, Characterization, and Release Study of Tacrolimus-Loaded Liquid Crystalline Nanoparticles

“…This is also in accordance with previous researchers' finding that lipolysis of monoolein in liquid crystalline nanoparticles causes structural changes in crystalline structure, leading to drug leakage. [27,28] Currently, a study is being conducted in our lab to see the additive effect of fatty acids to the composition of liquid crystalline nanoparticle on the release profile of tacrolimus. Figures 5A and 5B depict the percentage of tacrolimus remaining in the liquid crystalline nanoparticle formulations.…”

Thermomyces lanuginosus lipase-catalyzed hydrolysis of the lipid cubic liquid crystalline nanoparticles

“…We have previously studied lipase-catalyzed hydrolysis of cubic nanoparticles formed from GMO, which is the final step in the lipolysis of glycerol trioleate, leads to drastic changes in the liquid crystalline structure [13]. For that study we used lipase, TLL, conjugated to gold nanoparticles to visualize the enzyme location and the enzymatic digestion of lipid aggregates by means of cryogenic transmission electron microscopy.…”

Optimizing immobilization of avidin on surface-modified magnetic nanoparticles: characterization and application of protein-immobilized nanoparticles

“…Enzyme immobilization by covalent attachment to the organic layer of inorganic support surface via glutaraldehyde is one of the simplest and most gentle coupling methods in enzyme technology [9][10][11], but the activity of immobilized enzyme was reduced due to glutaraldehyde made up of a short carbon chain, which leads to steric hindrance between support and enzyme. On the other hand, the conjugation of the enzyme to the organic layer via the non-covalent biotin-avidin linking system is also an effective way [8,12,13]. In this case, there is a smaller set of biotin-binding sites used for binding to biotinylated protein and enzyme if the avidin is non-covalently immobilized on the surface of inorganic support and acts as a cross-linking agent, and this property may be a drawback.…”