Thermostability and irreversible activity loss of exoglucanase/xylanase Cex from Cellulomonas fimi.
The activity of the beta-1,4-glycanase Cex (EC 3.2.1.91) from Cellulomonas fimi is investigated in connection with its industrial application in cellulose hydrolysis and its potential use in cellosaccharide synthesis. Catalytic activity measurements as a function of temperature, complemented with differential scanning calorimetry (DSC) data, are used to characterize the thermostability of the protein and the influence of interdomain interactions. The data suggest that the enzyme is irreversibly deactivated in one of two possible ways: (1) through a low-temperature route characterized by first-order kinetics; or (2) through a high-temperature route characterized by an initial reversible step followed by an irreversible step. Melting temperatures (Tm) of Cex and p-33 (the isolated catalytic domain of Cex) as estimated by DSC are 64.2 and 64.0 degrees C, respectively, suggesting that the binding and catalytic domains of the protein fold independently. Kinetic parameters (Km, kcat, and kcat/Km) of Cex for the hydrolysis of p-nitrophenyl beta-D-cellobioside (pNPC) were determined at temperatures ranging from 15 to 80 degrees C. As demanded by reversible mass-action thermodynamics, the Tm of Cex in the presence of excess ligand as determined from activity-temperature data is ca. 66.55 degrees C, more than 2 degrees C higher than the Tm for Cex under ligand-free conditions. The effect of temperature on the rate constant has been determined using Arrhenius plots. Combined with irreversible deactivation half-life data and DSC data, the results are used to evaluate a model, based on a theory developed by Hei et al. (1993), for predicting the time-dependent activity and active-state stability of the protein under a range of potential operating conditions.
Nikolova
P V
PV
UBC Pulp and Paper Centre, Department of Chemical Engineering, University of British Columbia, Vancouver, Canada.
Creagh
A L
AL
Duff
S J
SJ
Haynes
C A
CA
D000465:Algorithms; D001426:Bacterial Proteins / Q000378:metabolism*; D002152:Calorimetry, Differential Scanning; D002480:Cellulase / Q000378:metabolism*; D043366:Cellulose 1,4-beta-Cellobiosidase; D004795:Enzyme Stability; D013696:Temperature
D001426:Bacterial Proteins; D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase