5-53: Understanding dynamics of cellulase adsorption on AFEX treated corn stover during the course of enzymatic hydrolysis

The adsorption of cellulases to the substrate is a prerequisite step for enzymatic hydrolysis of lignocellulosics. However, the mechanism of enzyme adsorption on crystalline cellulose is better understood than native lignocellulosics. In this study, high-throughput ‘Fast Protein Liquid Chromatography’ (FPLC) based enzyme separation and quantification based methods were developed.  Based on different isoelectric points of various cellulases such as cellobiohydrolase I (CBH I), cellobiohydrolase II (CBH II) and endo-glucanase I (EG I), individual enzymes could be eluted out separately from the column applying a linear conductivity gradient. Protein concentration could be correlated to the eluted peak area detected by UV absorption at 280 nm. Because this technique is non-destructive, eluted enzyme activity could also be measured.

Ammonia Fiber Expansion (AFEX) pretreated biomass has been found to improve enzyme accessibility. However, very little is understood about absorption dynamics of various cellulases and hemicellulases on AFEX treated biomass. In this study, AFEX treated corn stover was selected as the adsorption substrate. Cellulase (i.e. CBH I, CBH II, EG I) adsorption onto AFEX corn stover was studied and compared to untreated corn stover. Enzyme adsorption to lignin extracted from AFEX treated corn stover was also studied. The differential binding capacity of individual and multiple hydrolytic enzymes onto AFEX treated biomass was explored.

Investigating the enzyme adsorption behavior could help better understand the limiting factors affecting enzymatic hydrolysis. This work should also help in the rational design of synergistic enzyme cocktails that would maximize hydrolysis yields while reducing enzyme loading costs.