Tuning the catalytic activity of enzymes embedded in layer-by-layer assembled films

Abstract Immobilization of enzymes on supporting materials for biocatalysis is highly desired for industrial applications, because it endows the biocatalytic platform with significant advantages compared to free enzymes, e.g., easy separation, re-usability, and long-term stability. However, the possible denaturation of immobilized enzymes by the underlying substrate or crosslinking, and the limited amount of attached enzymes, often results in a low enzymatic activity. Here, we employ polyelectrolytes to assemble glucose oxidase in thick films in a layer-by-layer (LbL) way. By screening a large range of conditions, we show that the immobilized enzymes may retain their intrinsic structure and display a high activity when deposited in suitable conditions with the appropriate polycation. Moreover, the activity of formed multilayer enzyme films can be tuned depending on deposition conditions. We also demonstrate swelling of the film by the reaction medium by in-situ AFM measurements, with the film behaving as a hydrogel in which the enzymatic substrate can easily access the active site of enzymes immobilized all along the film. Hence, despite the complexity of protein adsorption in LbL films, conditions for stable growth of active enzymatic films of tunable activity can be found, offering a promising way to design highly active enzyme-immobilized catalytic platforms.