Multi-Enzyme Colocalization on Nanocarriers for Investigating Biosynthesis

Many cascaded biological reactions are catalyzed by enzymes that exist as multi-enzyme complexes. The spatial colocalization of enzymes in these complexes increases the reaction efficiency by ensuring that the reactive intermediates have rapid access to neighboring active sites and maintaining high local concentrations. Although some work has been done on colocalizing model enzymes, there is a lack of knowledge regarding less robust enzymes. Inspired by nature, we immobilized and colocalized two diterpene synthases (DTS) that catalyze the production of sclareol, a valuable fragrance prized in the perfume industry, onto polymeric and nonpolymeric nanoparticles (NPs). Colocalization of DTS enzymes onto NPs can increase the reaction efficiency by producing local environments that facilitate the catalysis of sclareol. The immobilization of these enzymes onto a NP can also improve their stability. Our strategy for immobilization was designed for the binding of histidine-tagged enzymes on to nickel nitrilotriacetic acid-functionalized NPs. We investigated the binding efficiency and retained catalytic activity of the enzyme-nanoparticle conjugates for silica NPs (SiNPs), polystyrene NPs, and gold NPs. We achieved significant binding for SiNPs, but there was little retained catalytic activity (1.1%). Polystyrene NPs also bound a substantial amount of enzymes, but sclareol production could not be determined because these NPs interfered with our hexane extraction process. Gold nanoparticles did not disrupt the activity of the enzyme, but the high cost of this material made it prohibitively expensive to procure quantities that would load a substantial amount of enzymes. An ideal nanocarrier will be one that yields a high binding capacity while retaining the catalytic activity of the loaded enzymes and should be amenable to quantitative assays and extractions.