Combinatorial Approaches for Effective Design, Synthesis, and Optimization of Enzyme-Based Conjugates

Abstract

The specificity and efficiency with which enzymes catalyze selective chemical reactions far exceeds the performance of traditional heterogeneous catalysts that are predominant in industrial applications such as conversion of commodity chemicals to value-added products, fuel cells, and petroleum refinement. Moreover, biocatalysts exhibit exceptionally high product turnover at ambient conditions with little health and environmental burden. These advantageous qualities have led to the prolific use of enzyme catalysis in pharmaceutical, detergents, and food preservation industries wherein their use has greatly reduced waste generation, Unfortunately, the full slate of benefits that enzymes can impart to a broader range of chemical processes is severely hindered by lack of enzyme reusability and/or denaturation of native enzyme structure in industrially relevant conditions, i.e., high temperatures, turbulent flow regimes, non-aqueous solvents. Enzyme immobilization—the interfacing of biocatalysts with materials possessing properties of interest—is a well-studied strategy to address the shortcomings of free enzyme catalysis. Notably, immobilized enzymes demonstrate improved operability in non-physiological reaction conditions, lengthened shelf lives, and can be separated from reaction mixtures for subsequent reuse. Enzyme immobilization itself is however not a universal solution to the shortcomings that characteristic of free enzyme catalysis. This is because the immobilization of target enzymes often incurs mass transfer limitations, additional costs of materials, and loss of biocatalyst activity that can be prohibitive in the viable implementation. Thus, facile enzyme immobilization techniques require intensive investigation into the interactions between enzymes and carrier/scaffold materials that are not only determinant in the feasibility of immobilized enzymes applications but are also enlightening to the ongoing efforts to establish meaningful relationships between enzyme structure and function that will preserve extended biofunctionality in a variety of synthetic environments. Herein, we hypothesize that a strategy encompassing both laboratory-scale and computational work should be used to more fully understand the chemical and physical interactions that occur between an immobilized enzyme and different immobilization supports and thereby critically assess the interface between the two can be controlled to develop improved, user-controlled strategies for robust, efficient immobilized biocatalysis. To demonstrate our hypothesis, we have implemented our strategy on two material systems that are promising in future enzyme immobilization applications. The first material is a hyaluronic acid (HYA)-based polymer network that we have chemically modified to tailor its physicochemical properties for use as an enzyme immobilization platform for biomedical applications. HYA-based hydrogels were synthesized and characterized via techniques like Fourier transform infrared (FTIR) spectroscopy, thermogravimetric (TG) analysis, nuclear magnetic