Monday, May 2, 2011
Grand Ballroom C-D, 2nd fl (Sheraton Seattle)
Cellulase binding to insoluble lignocellulosics is the essential first step for deconstruction of biomass to soluble sugars. However, the role of substrate characteristics (e.g., cellulose crystallinity, lignin content) on enzyme binding is still far from understood. We have used a recently developed high-throughput fast flow liquid chromatography (HT-FPLC) method to simultaneously quantify endoglucanase I (EG I), cellobiohydrolase I (CBH I) and cellobiohydrolase II (CBH II) binding to various insoluble cellulosic substrates. The binding affinity of cellulases during the course of enzymatic hydrolysis (0-48 hours) was monitored for various cellulose allomorphs: native crystalline cellulose I, cellulose III, cellulose II and amorphous cellulose (AC). The relative digestibility ranking was AC>cellulose III>cellulose II>cellulose I. However, AC had the highest initial cellulase binding capacity while cellulose III had the lowest. After near-theoretical substrate solubilization, most of the cellulase was recoverable from the supernatant with the exception of CBH II. Similar binding studies were performed for pretreated lignocellulosic biomass; namely, ammonia fiber expansion (AFEX-CS), dilute acid (ACID-CS) and ionic liquid (IL-CS) pretreated corn stover. For AFEX-CS, the initial cellulase binding was >80% of total amount added. However, after 48 hours of near-theoretical digestion, only around 50% of CBH I, 28% of CBH II and 19% of EG I were detected in the supernatant. Similar results were found for ACID-CS. We find that lignin was largely responsible for unproductive cellulase binding. These results provide insight into mechanisms of cellulase synergism, productive/unproductive cellulase binding and the role of pretreatment on enzyme accessibility for lignocellulosic biomass.