Immobilized Enzyme System for Lignocellulosic Biomass Saccharification

The overall objective of this research is to enhance the bioactivity of cellulase enzymes as well as the stability of the enzymes to increase it reusability through immobilization of the enzymes in the microenvironment of porous inorganic-organic hybrid sol-gel polymers. Recently, we have discovered that the microenvironment of these sol-gel polymers markedly increases the bioactivity of immobilized enzymes. Using these polymers for the confinement of the enzymes or as supports to covalently attach them will allow us to explore the effects to these two different approaches to immobilization on the bioactivity and reusability of cellulase enzymes. We have prepared polymers in which β-glucosidase, a cellulase enzyme, has been covalently immobilized onto sol-gel polymers and integrated with capillary electrochromatography as a separation scheme downstream from the immobilized enzyme. At room temperature, the immobilized enzyme in a sol-gel polymer with high porosity was able to partially hydrolyze D-(-)-salicin, our model substrate, to saligenin in a 5-minute period, while a room-temperature hydrolysis of salicin with the enzyme did not produce saligenin. Background The most critical step in the cellulose-to-ethanol bioconversion appears to be saccharification, the enzyme hydrolysis of cellulose to produce fermentable sugars that can yield ethanol. The high cost of cellulases makes it desirable to be able to reuse the enzymes and increase their bioactivity. Recently, we have discovered that confined enzymes that have been immobilized in microenvironments have markedly increased bioactivity. Specifically, we have reported that pepsin immobilized in a porous inorganic-organic hybrid sol-gel monolith exhibited a 700-fold increase in bioactivity and similarly encapsulated trypsin was found to have enhanced stability and bioactivity. Covalent attachment of trypsin on a similar hybrid sol-gel polymer resulted in a 2000-fold increase in the bioactivity of the enzyme for the hydrolysis of benzoyl-L-arginine ethyl ester, a substrate commonly used to determine the bioactivity of trypsin. These porous hybrid sol-gel polymer monoliths indeed appear to be good candidates for the confinement of enzymes, resulting in increased enzymatic hydrolysis at room temperature. Fundamental understanding of how confined, immobilized enzymes are able to increase their activity is presently lacking, although several hypotheses exist. The purpose of this study will be to elucidate this enzymatic enhancement mechanism and to apply it to the pressing need of converting biomass to biofuel. Results Our study of confined enzymes starts with β-glucosidase, one of the cellulase enzymes. We have prepared immobilized β-glucosidase in a porous sol-gel polymer monolith following a previously developed protocol for enzyme immobilization. The immobilized enzyme-sol-gel