Nanostructured Polymeric Materials and Their Implementation in Enhancing Biocatalytic Processes

Polymer science has played a pivotal role in the development of nanotechnology. Self-assembly of polymeric materials at the molecular-level enables the fabrication of periodic arrays of nanostructures with various geometries and sizes, which present exciting opportunities in fabrication of nanoscale devices. However, self-assembled structures are often too regular for use in fabricating complex devices. Therefore, a simple layer-by-layer method was developed to allow for quantitative control over both size and shape of equilibrium nanostructures in self-assembled block copolymer thin films. The use of photolithography imparted spatial control over the self-assembly process.Such self-assembly can result in structures approaching the size of single biomacromolecules, potentially enabling the precise placement of individual enzymes. Using these nanostructured thin films as inspiration, the effect of nanopatterning enzymes for multi-step biocatalysis was explored numerically by developing a kinetic Monte Carol simulation. Molecular trajectories of the reaction species as well as turnover frequency of individual enzymes on the surface were tracked under diffusion-limited and reaction-limited conditions. Interestingly, these simulations revealed that enzyme density and arrangement have little impact on overall activity of the multi-enzyme cascade reaction.Given these results, we turned our attention to improving enzymatic activity by covalent modification of the enzyme with polymeric materials. This covalent modification holds tremendous promise as an approach to tune the molecular-level interactions between enzymes and their solvent environments. Enzymes modified with highly soluble polymers had greatly improved solubility in an ionic liquid. This correlated with increases of up to 19-fold in enzyme activity. However, because the preparation and purification of enzymes can be costly, the loss of recyclability of the newly homogeneous enzyme-polymer conjugates was undesirable. By utilizing a responsive polymeric material, the miscibility of the enzyme can be altered adaptively. Specifically, thermodynamic interactions between the enzyme-polymer conjugate and solvent were varied as a function of temperature by utilizing a thermoresponsive polymer. When recycled via sequential dissolution and precipitation, the enzyme did not lose any activity. This approach enables the benefits of both increased activity of homogeneous biocatalysis and improved processability of heterogeneous biocatalysis in non-native solvents

Role of Dimension and Spatial Arrangement on the Activity of Biocatalytic Cascade Reactions on Scaffolds

Despite broad interest in engineering enzyme cascades on surfaces (i.e., for multistep biocatalysis, enzyme-mediated electrocatalysis, biosensing, and synthetic biology), there is a fundamental gap in understanding how the local density and spatial arrangement of enzymes affect overall activity. In this work, the dependence of the overall activity of a cascade reaction on the geometric arrangement and density of enzymes immobilized on a two-dimensional scaffold was elucidated using kinetic Monte Carlo simulations. Simulations were specifically used to track the molecular trajectories of the reaction species and to investigate the turnover frequency of individual enzymes on the surface under diffusion-limited and reaction-limited conditions for random, linear striped, and hexagonal arrangements of the enzymes. Interestingly, the simulation results showed that, under diffusion-limited conditions, the overall cascade activity was only weakly dependent on spatial arrangement and, specifically, nearest-neighbor distance for high enzyme surface coverages. This dependence becomes negligible for reaction-limited conditions, implying that the spatial arrangement has only a minimal impact on cascade activity for the length scales studied here, which has important practical implications. These results suggest that, at short length scales (i.e., sub 10 nm dimensions) and high enzyme densities, sophisticated approaches for controlling enzyme spatial arrangement have little benefit over random immobilization. Moreover, our findings suggest that engineering artificial cascades with enhanced activity will likely require direct molecular channeling rather than a reliance on free molecular diffusion