Monday, April 30, 2012
Napoleon Ballroom C-D, 3rd fl (Sheraton New Orleans)
Enzymatic hydrolysis of cellulose is a key step in the bioconversion of lignocellulosic biomass to biofuels. The exact mechanism of hydrolysis of several key enzymes has been extensively studied, however, many questions remain unanswered. The binding of cellulases to cellulose has always been thought to be the crucial step preceding efficient hydrolysis. Albeit recent work from our lab has shown that crystalline cellulose III gave a higher rate of hydrolysis despite reduced cellulase binding affinity relative to native cellulose I (Chundawat et al., 2011. JACS 133, 11163-11174). This finding highlights our lack of understanding of cellulase interaction with non-conventional cellulosic allomorphs like cellulose III. In this study, the binding kinetics of the cellulose-binding module (CBM) of cellobiohydrolase I (CBH I) was evaluated using isothermal titration calorimetry (ITC) for native cellulose I and cellulose III (derived from ammonia pretreated bacterial microcrystalline cellulose or BMCC). CBH I was isolated from a Trichoderma reesei enzyme broth using fast protein liquid chromatography (FPLC). The enzyme was partially proteolyzed using papain and the CBM was isolated for further purification using FPLC. Isolated peptide structure was elucidated by liquid chromatography tandem mass spectrometry. ITC experiments were carried out using the isolated CBM on the proposed substrates to explore the thermodynamics of CBM-cellulose interaction. Additionally, since it is suspected that glycosylation of CBH I may impact its binding, protein deglycosylation was carried to study impact of glycosylation on cellulase-cellulose interaction.