7-35: The determination of individual enzyme adsorption profiles during the hydrolysis of pretreated lignocellulosic substrate by specific immunoassays

One way to reduce the cost of the enzyme hydrolysis step of a biomass-to-sugars process is to try to reuse the robust and stable enzymes that are found in a typical cellulase enzyme mixture. However, to do this, we need to have a better understanding of how the enzymes adsorb, distribute, and interact with the substrate if we are to both improve the efficiency of enzymatic hydrolysis and develop an effective enzyme recycling strategy. In previous work, we followed the adsorption and activity profiles of 6 specific enzymes present in a commercial cellulase preparation during the hydrolysis of steam-pretreated corn stover (SPCS). A variety of techniques including zymograms, gel electrophoresis, enzyme activity assays and mass spectrometry were used to provide a semi-quantitative measurement of the distribution of each of the enzymes. However, this proved to be laborious, and the qualitative nature of the assays highlighted the need to develop a more refined assay that could specifically quantify individual enzyme components during hydrolysis and enzyme recycle. It has been shown that an antibody’s ability to specifically recognize a target antigen can provide a direct and highly specific tool to quantify the different components in an enzyme mixture. We have developed an antibody-based assay to follow and quantify individual enzyme components during the hydrolysis of pretreated biomass substrates. This high throughput assay proved to be direct, sensitive, quantitative, and specific for the target enzymes while encountering minimum interference from other components present in the lignocellulosic substrate.