(491f) The Binding Properties of Cellulases On Allomorphs Cellulose and Pretreated Biomass During Enzymatic Hydrolysis

Authors: 
Gao, D., Michigan State University
Chundawat, S., Michigan State University
Balan, V., Great Lakes Bioenergy Center, Michigan State University
Dale, B., Michigan State University


The depolymerization of cellulose to monomeric glucose is the key step for cellulosic ethanol biorefinery process. Cellulases binding on biomass is the essential first step for effective breakdown of biomass to sugars. A new fast flow liquid chromatography (FPLC) based method has been developed to quantify endoglucanase I (EG I, GH family 7B), cellobiohydrolase I (CBH I, GH family 7A) and cellobiohydrolase II (CBH II, GH family 6A) individually in the heterogeneous reaction environment.

The binding level of individual enzymes during 48 hours hydrolysis was studied on different cellulose allomorphs: micro crystal cellulose Avicel (cellulose Iβ), liquid ammonia treated cellulose (cellulose III), sodium hydroxide treated cellulose (cellulose II) and phosphoric acid swollen amorphous cellulose (AC). The digestibility rank is AC>cellulose III>cellulose II>cellulose I. However, the AC has the highest initial binding capacity while cellulose III has the lowest. After the substrate solubilized, most of the cellulase returned to supernatant. But CBH II is less stable. Only around 60% CBH II can be recovered.

The time course binding studies were also performed under pretreated biomass. Ammonia Fiber Expansion (AFEX) treated corn stover (CS), dilute acid treated CS and ionic liquid pretreated CS are compared. The results show the existence of lignin is responsible for a significant amount of unproductive binding. For AFEX-CS, the initial binding level for those cellulases are >80%. However, after 48 hours digestion, only around 50% of CBH I, 28% of CBH II and 19% of EG I are detected in the supernatant. The remaining cellulases are believed to be bound unproductively onto lignin. Similar results were found on ACID-CS. The results are critical for improving our understanding of enzyme synergism, productive/unproductive enzyme binding and the role of pretreatment on enzyme accessibility to lignocellulosic plant cell walls, as well as help to engineer novel low unproductive binding enzymes and develop an economic enzymes recycle system.