Surface kinetics for cooperative fungal cellulase digestion of cellulose from quartz crystal microgravimetry.
The kinetic behavior of aqueous cellulase on insoluble cellulose is best quantified through surface-based assays on a well-defined cellulose substrate of known area. We use a quartz crystal microbalance (QCM) to measure the activity of binary mixtures of Trichoderma longibrachiatum cellobiohydrolase I (Cel7A) and endoglucanase I (Cel7B) on spin-coated cellulose films. By extending a previous surface kinetic model for cellulase activity, we obtain rate constants for competitive adsorption of Cel7A and Cel7B, their irreversible binding, their complexation with the cellulose surface, and their cooperative cellulolytic activity. The activity of the two cellulases is linked through the formation of cellulose chain ends by Cel7B that provide complexation sites from which Cel7A effects cellulose chain scission. Although the rate-limiting step in Cel7A activity is complexation, Cel7B activity is limited by adsorption to the cellulose surface. A 2:1 bulk mass ratio of aqueous Cel7A:Cel7B, corresponding to a 4:1 surface mass ratio, effects the greatest rate of cellulose degradation across a range of cellulase concentrations at 25 °C. We find that surface chain-end concentration is a major predictor of Cel7A activity. Disruption of the hydrogen-bonding structure of cellulose by Cel7B enhances the activity of Cel7A on the cellulose surface.
Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA 94720-1462, USA.
S A
SA
Maurer
N W
NW
Brady
N P
NP
Fajardo
C J
CJ
Radke
D002482:Cellulose / Q000378:metabolism*; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000378:metabolism*; D007700:Kinetics; D058525:Quartz Crystal Microbalance Techniques; D014242:Trichoderma / Q000201:enzymology*
D002482:Cellulose; D043366:Cellulose 1,4-beta-Cellobiosidase