March 8-13, 2009
Cellulose, the major structural component of plant cell walls, is a homopolymer of β-1,4-linked glucose residues. As cellulose is the most abundant biopolymer on Earth comprising approx. 50% of the bioshpere, it has attracted renewed interest as a potential source of energy through its biodegradation and fermentation to biofuels. The biodegradation of cellulose involves the concerted action of three types of enzymes, cellulases (EC 184.108.40.206, endo-β-1,4-glucanases), cellobiohydrolases (EC 220.127.116.11; cellulose 1,4-β-cellobiosidase), and β-glucosidases (EC 18.104.22.168; β-d-glucoside glucohydrolase). The former two classes of enzymes function to hydrolyze insoluble cellulose into soluble oligosaccharides which then serve as substrates for β-glucosidases to release free glucose. In many cases, these enzymes are multi-modular, being comprised of distinct catalytic and carbohydrate-binding modules (CBMs). The CBMS appear to aid in both the adsorption of the enzymes to the insoluble cellulose substrate and the destabilization of the hydrogen-bonding network within the crystalline substrate. An understanding of this latter process is extremely important because it has been demonstrated that binding of the enzymes to the insoluble cellulose represents the rate-limiting step in its hydrolysis. To this end, we have developed a protocol for the direct and real-time observation of cellulose biodegradation by atomic force microscopy (AFM).
Working electrodes for AFM experiments consisted of a 200 nm thick gold film vapor deposited onto a glass slide pre-treated with a deposition of a 2 nm thick layer of chromium. After annealing in a muffle furnace at 700°C for 60 s, the slides were treated with thioglucose to provide a highly-ordered monolayer of hydrophilic glucose for the stable adsorption of cellulose. Thin films of bacterial microcrystalline cellulose on these electrodes were prepared using the Langmuir-Blodgett technique. Optimized conditions were established to involve a dispersion of a 2 mg/ml suspension of cellulose in methanol/chloroform (1:5) on aqueous phosphate buffer using a compression of 5 mN/m. With this protocol, drying of the cellulose film thereby precluding any associated structural alterations.
AFM images were captured using a Pico SPM Microscope with AFMS 182 scanner and Pico-scan 5.2 software system using silicon nitride tips which had a nominal spring constant of 0.06 N m-1 for contact mode, and magnetically coated silicon tips for MAC mode. Under these conditions, the diameters of the microfibrils in a 50 nm fiber were observed to be 3 - 4 nm, which is smaller than the 7.5 nm previously reported by others. Homogeneous samples of the cellulase CenA from the bacterium Cellulomonas fimi were introduced into the liquid cell through capillary ports for the in situ imaging of cellulose disruption and hydrolysis. This activity was monitored over the course of 19 hours and initial evidence of degradation of the fibers was observed within three minutes of enzyme addition. In addition, details of the process of fiber fraying could be readily discerned. Genetic engineering was used to provide a mutant form of CenA involving a replacement of its catalytic aspartate nucleophile with alanine. Studies with this catalytically inactive enzyme derivative permit the analysis of cellulose fibril destabilization prior to hydrolysis.
Anthony Clarke, Amanda Quirk, Jacek Lipkowski, Darrell Cockburn, and Dan Glickman, "REAL-TIME OBSERVATION OF CELLULOSE BIODEGRADATION BY ATOMIC FORCE MICROSCOPY" in "Bioenergy - II: Fuels and Chemicals from Renewable Resources", Dr. Cedric Briens, ICFAR, University of Western Ontario, Canada; Dr. Franco Berruti, ICFAR, University of Western Ontario, Canada; Dr. Muthanna Al-Dahhan, Washington University, USA Eds, ECI Symposium Series, (2009). http://dc.engconfintl.org/bioenergy_ii/8