Novel polymer-assisted organic-inorganic hybrid nanoflowers for cascade biocatalysis

Abstract

ABSTRACT (250-300 words) Background: There is increasing interest towards multienzyme complexes, in which several enzymes are co-immobilized on the same support used to catalyze cascade reactions. Major advantage of these complexes is that the enzymes´ active sites are in close proximity, minimizing the diffusion of the intermediates thereby enhancing the overall reaction efficiency. The compartmentalization of the multienzyme complex is crucial, thus new enzyme organization and supports are being developed. Objective: Design of novel multienzyme polymer-assisted organic/inorganic nanoflower complexes that are reusable and act as effective cascade biocatalysts. They combine the resistance and robustness of polyamide-6 (PA6) microparticles (MP) with the improved activity of the enzymes organized in nanoflover (NF) structures. Methods: PA6 MP were synthesized by activated anionic ring opening polymerization in solution and used as carriers of hybrid bienzyme nanoflowers containing glucose oxidase (GOx) and horseradish peroxidase (HRP). Four complexes with different co-localization and compartmentalization of the two enzymes on the PA carrier (PANF) were made and analyzed morphologically. Their catalytic activity was assessed in the cascade reaction for glucose detection and the kinetic parameters were determined. In addition, the reusability, and the storage stability of the cascade PANF catalysts were also studied. Results: The SEM micrographs proved an almost spherical morphology of the NF entities with average diameters of 5-15 µm and differently nanostructured petals. The effect of the enzymes´ co-localization in PANF on the kinetics of the GOx/HRP catalyzed cascade reaction was studied. Some PANF displayed relative activities being three times higher than the free enzyme dyad. The PANF activities correlate well with the Michaelis-Menten kinetic parameters Vmax and Km. All PANF remain active for up to 6 consecutive operational cycles, furthermore, being also more stable than the free dyad. They displayed good storage stability at 4 ºC losing only 2-15% of their initial activity after 60 days. Conclusion: The multienzyme PANF complexes are significantly more active and more stable than the free enzyme dyad or the classic nanoflowers which can open the way of their biotechnological application.