Interfacing Enzymes and Polymer Brushes: Tailoring Interactions for Optimizing Immobilized Biocatalysis

Sánchez-Morán, Héctor

Graduate Thesis Or Dissertation

Interfacing Enzymes and Polymer Brushes: Tailoring Interactions for Optimizing Immobilized Biocatalysis Public Deposited

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Citeable URL: https://scholar.colorado.edu/concern/graduate_thesis_or_dissertations/nv9354682

Abstract

Enzyme immobilization is a technique consisting in fixing an enzyme molecule to a solid-liquid interface, which provides enzymes with several advantages like recyclability or, in some cases, stabilization. However, the molecular mechanisms behind stabilization are poorly understood, causing some enzymes to be destabilized by immobilization materials without understanding the underlying cause. In fact, growing evidence of enzymes being destabilized by conventional or “gold-standard” materials suggests that there is no such thing as a universally stabilizing material for enzyme immobilization, requiring a paradigm shift and the development of new knowledge to understand and leverage enzyme-material interactions which result in enzyme stabilization.

In this work, we have explored the use of surface-grafted random copolymer brushes as immobilization supports for enzymes. These supports are unique in that they are highly dynamic in molecular length scales, which enables transient interactions between enzymes and polymers. It is thought that moieties in the polymer layer can transiently self-assemble with regions of matching properties in the enzyme surface, which ultimately results in stabilizing interactions with potential to promote the folded and active state of enzymes. To understand mechanisms of interaction between enzymes and polymer brushes, we explored the immobilization of several enzymes on polymer brushes covering a wide range of hydrophilicity. Interestingly, several of these enzymes, despite similar in their function, displayed differential behavior on supports of different hydrophobicity. By studying the surface characteristics of the immobilized enzymes, and comparing them with characteristics of immobilization supports, we devised a general design rule to enable the prediction of immobilized enzymes on demand. Then, we explored the effect of other types of enzyme-material interactions by incorporating novel aromatic-containing monomers on immobilization supports. Following studies of the impact of these moieties on the activity of immobilized enzyme, we employed single molecule FRET microscopy studies to unveil mechanisms of interaction between enzymes and novel aromatic-containing materials. Lastly, we developed a tool for high-throughput screening of polymeric supports for immobilized enzymes, to expedite the exploration and discovery of complex copolymeric supports for enzyme immobilization. Ultimately, this work represents a set of versatile tools to help enzyme engineers immobilize enzymes of their interest, which could have important implications in their use as immobilized biocatalysts for industrial applications.

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