(485l) Performance Analysis of a Free and Polymer-Conjugated Multi-Enzyme System for Ethanol Metabolism

Our design of a multiple enzyme system, composed of alcohol (ADH), aldehyde (ALDH) and lactic (LDH) dehydrogenases, has previously shown promise as a kinetically effective mechanism to improve alcohol metabolism, while also preventing toxin buildup, in the human body. Multi-enzyme experimental data, combined with simulation modeling, has provided new information on controlling kinetic terms of the defining enzyme rate equations. This data has revealed favorable enzyme ratios for achieving best conversions of ethanol while preventing concentration barriers brought on by substrate inhibition in both ALDH and LDH as well as product inhibition among ADH and ALDH. Further modeling also demonstrates that efficient conversion of ethanol to non-toxic acetate can still be achieved under complex initial concentrations of various reactants and products.

Covalent attachment of the three enzymes within a poly(MAA-co-PEG200MA-co-PEG200DMA) pH-sensitive network has been accomplished via UV free-radical polymerization following functionalization of the enzyme amino groups with acryloyl chloride. Individual enzyme activity changes during each fabrication step were monitored. Conversion analysis of key system components, ethanol, lactate, and NADH, in the final three-enzyme polymer show similar activity retention, up to 26%, of each enzyme in gel form when compared to free enzyme situations. Stability of the polymer-conjugated enzymes to various pH cycles establishes good protective capability of the hydrogel matrix against harsh environments. Additionally, multi-enzyme hydrogel microparticles were created to evaluate diffusion transport limitations, and studies have indicated ample transport in thin film and particle gels. This novel three enzyme system with concerted activity within a biocompatible network emphasizes the tremendous, emerging potential of multi-enzyme biofunctional gels.