Increase of catalytic activity of lipase towards olive oil by Langmuir-film immobilization of lipase

Proteins represent versatile building blocks for realization of nanostructured materials to be applied in nanobiotechnology. In the present work, the Langmuir–Blodgett technique was utilized to develop nanobiodevices based on protein molecules. Particularly, lipase thin films were fabricated and characterized, with characterization performed in order to optimize the working parameters. As the first step the protein films were studied at the air–water interface and then transferred onto a solid support for further characterization. The films were characterized by different techniques such as UV–Vis spectroscopy, nanogravimetry, atomic force microscopy, and biochemical assays. Catalytic activity of lipase characterized by the maximal reaction rate found to increase over 10 times as a result of inclusion into LB films, while the substrate binding characterized by the Michaelis constant remain unchanged. Catalytic activity per mole of enzyme was found to increase with the increased number of LB layers up to five, and then decrease at 10, while the surface coverage ranged from 70% to 100% from 1 to 10 layers of lipase. This study exploits the possibility to employ LB based protein structures to use in biocatalysis, exemplified by lipase, which is known as an interfacially-activated enzyme, with olive oil as substrate, when lipase should already be in the maximally active state even without a film. We show, however, that it was possible to form even more active lipase nanostructures by the Langmuir–Blodgett technique at the air–water interface, proving that Langmuir-film provides a better catalytic effect in lipase than a mere oil–water boundary.