(704e) Design of Nanoparticle-Based Platforms for Multi-Enzyme Co-Localization

In this research, we have designed nanoparticle-based platforms to co-localize multi-enzyme complexes with reactive intermediates. An example of these multi-enzyme systems is anthocyanidin synthase (ANS) and anthocyanidin reductase (ANR), where the two enzymes need to be co-localized in order to rapidly transport the highly reactive intermediate cyanidin from ANS to ANR to maximize the production of flavon-3-ols, a powerful anti-oxidant, which are beneficial for plant and human health. As a first step, glucose oxidase (GOX) and horseradish peroxidase (HRP) were used as model enzymes to demonstrate co-localization of multiple enzymes on polymeric nanoparticles. Hydrogen peroxide formed in the first reaction catalyzed by GOX is consumed by HRP in the second reaction in the presence of N-acetyl-3,7-dihydroxyphenoxazine (Amplex Red), to yield a specific fluorescent product, resorufin. To optimize the enzyme attachment, covalent binding, streptavidin-biotin coupling and polyethylene glycol (PEG) spacer linkage were evaluated and compared by immobilizing HRP on carboxylated polystyrene nanoparticles. The blank, functionalized, and enzyme-attached nanoparticles were characterized by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), and dynamic light scattering (DLS). The enzyme kinetics were evaluated before and after the attachment. The results showed that the PEG spacer linkage and streptavidin-biotin coupling were comparable, and both were superior to covalent binding in terms of maintaining the stability and activity of HRP. Furthermore, different spacer lengths of PEG were investigated. The GOX and HRP were co-localized on the nanoparticles through PEG spacer linkage and the two step reaction kinetics were evaluated and compared with single-enzyme complexes and free enzymes. Based on the optimization carried out with the model system, ANS and ANR were co-localized on the nanoparticles through appropriate PEG spacer attachments. This study demonstrates a simple and effective way for multiple enzyme co-localization to mimic the efficient multi-enzyme complexes that widely exist in Nature.