Catalytic Enhancement of Enzymatic Pathways on a Nanoparticle Surface

Enzymes are of tremendous interest for the industrial production of numerous products due to their incredible ability to carry out chemical transformations that are highly specific and often quite difficult, if not impossible to replicate using synthetic means.  However, their use has been largely limited due to their instability, purification costs, and difficulty in separating the enzymes from products for re-use.  Efforts at enhancing the performance of enzymes and their viability in industrial syntheses have often focused on the areas of protein engineering and enzyme immobilization.  Protein engineering, while successful in certain cases, is often quite difficult and often produces only modest improvements in catalytic activity or preferences for somewhat analogous substrates.  Immobilization of enzymes on the other hand often leads to decreased catalytic activity relative to free enzymes, thus limiting its overall effectiveness.  Recently our lab has demonstrated that immobilization of enzymes on quantum dot (QD) surfaces is often able to enhance the activity of enzymes, including a “perfect” diffusion-limited enzyme.  Contrary to many other methods of enzyme immobilization, this technique is quite facile, requiring only a histidine tag to attach the protein to the inorganic surface.  We have now investigated the effect of QD immobilization within a multienzyme cascade, using freely diffusing glucose oxidase (GOX) coupled to QD-immobilized horseradish peroxidase (HRP).  The immobilized HRP displays enhanced enzyme kinetics, and appears to be activated either by substrate accumulation near the QD surface or improvement in the catalytic properties of HRP. By optimizing the ratio of GOX to HRP, we were able to preserve the catalytic enhancement of HRP, demonstrating that the presence of additional catalytically-coupled solution phase enzymes with the QD immobilized enzyme doesn’t appear to interfere with the QD-dependent enzymatic enhancement.  We are now extending the power of enzyme immobilization on QDs by binding multiple enzymes from glycolysis to a single surface to determine whether we can further enhance the activity of the system by minimizing diffusion between enzymes.  As part of this study we are also investigating whether QD-dependent enzymatic enhancement of a histidine-tagged protein can be achieved without first purifying the enzyme from the cell lysate.  Additional efforts are underway to attempt to organize QDs on larger inert surfaces in an attempt to create additional surface area for enzyme binding and colocalization.  It is our hope that these efforts will lead to a widely applicable method for immobilizing enzymes while preserving and enhancing their activity within complex enzymatic pathways.  By doing so, we hope to create better “green” catalysts that can be used for the industrial synthesis of numerous products.