Multi-Enzyme System for the Rapid Metabolism of Ethanol: Kinetics and Conjugation Within pH-Sensitive Hydrogel

Abstract

Biohybrid materials, or materials utilizing biological molecules in their rational design, are prime therapeutic carrier candidates to perform detoxification processes. Specifically, catabolic processing of alcohol is a significant component of the glycolytic pathway to yield energy from alcohol. Moreover, the process is crucial to preventing toxic buildup of ethanol and its unstable, free-radical forming byproduct acetaldehyde. Our work introduces a biomimetic hybrid hydrogel structure capable of facilitating efficient ethanol conversion through enzymes delivered to the body. A multi enzyme system, composed of alcohol (ADH) and aldehyde (ALDH) dehydrogenases for conversion of ethanol to non-toxic acetate coupled with lactate dehydrogenase (LDH) for cofactor recycling, was kinetically defined by our research group with experimental data and simulations to provide rapid conversion of ethanol. The system at desired enzyme concentrations overcomes substrate and product inhibitions to metabolize ethanol at rates seven times faster than the human body. A system involving the covalent attachment of ADH to a poly(MAA-co-PEG200MA-co-PEG200DMA) pH-sensitive network via UV free-radical polymerization following functionalization of the enzyme amino groups with acryloyl chloride was also thoroughly studied. After monitoring individual enzyme activity during each fabrication step, the enzyme-enhanced polymer system shows activity retentions up to 26%. Stability testing of the polymer-conjugated enzymes under pH cycles (pH 4.0-7.5) demonstrates minor loss of activity and establishes protective capability of the hydrogel matrix against proteolytically harsh environments. This novel enzyme system and the demonstration of enzyme stability within a biocompatible hydrogel network emphasize the tremendous, emerging potential of multi-enzyme biofunctional gels.