(12d) Combinatorial Experimental and Computational Approaches Facilitate Implementation of Amphiphilic Hydrogels for User-Controlled Enzyme Immobilization

Biocatalysis holds several key advantages over traditional chemical catalysis—high substrate specificity, high product selectivity, high degree of reactivity at ambient conditions, low toxicity, and minimal environmental impact—that have eventuated the implementation of biocatalysts in industrial applications spanning from pharmaceuticals, to biofuel, food and beverage, and detergents sectors. However, the entire slate of benefits offered by biocatalysis has yet to be fully realized in industrial settings due to loss of biocatalyst activity as a result of temporal instability of relevant enzymes and enzyme denaturation in non-physiological conditions, e.g., high temperatures, turbulent flow regimes, toxic industrial solvents etc.

Advances in enzyme immobilization onto novel materials for specific end applications have been shown to improve enzyme stability, expand enzyme operating conditions, and provide criteria for facile separation and reuse of enzymes industrially implemented. However, enzyme immobilization itself introduces drawbacks—mass transfer limitations, reduced catalytic efficiency, additional costs of immobilization materials—that necessitate the optimization of biocatalysts immobilized in inexpensive, sustainably derived materials.

We propose a design strategy to allow for the immobilization of model enzyme glucose oxidase (GOx) to amphiphilic hydrogels derived from naturally occurring biopolymer hyaluronic acid to thus mitigate both loss of catalytic efficiency over time or due to denaturation in non-native conditions. Kinetic studies of the hydrogel-immobilized enzyme were conducted to determine apparent interfacial kinetic parameters of GOx to elucidate catalytic efficiency of the immobilized enzyme relative to free one while also evaluating effectiveness of immobilization. Further, molecular dynamics studies were used to probe self-assembly properties of the amphiphilic hydrogel and to reveal the chemical and physical interactions occurring between the hydrogel and GOx that do not only control the interface between two systems but further, affect their overall functionality. The change in dynamics of hydrogel self-assembly relative to varied synthesis conditions was used to optimize composite efficiency of the immobilized biocatalyst, i.e., maximal enzyme loading to the hydrogel and thus improve enzyme stability and functionality. The enhanced suitability of GOx-containing hydrogels over free GOx in industrial settings was examined via both force measurements to assess mechanical stability of the system as well as kinetic studies in non-physiological conditions. The results demonstrate we developed a feasible strategy for the controlled immobilization of biocatalysts in synthetic environment using inexpensive, renewable resources with extended end uses in biosensor, food preservative, or chemical production applications.