Surface Diffusion of Cellulases and Their Isolated Binding Domains on Cellulose*
- From the Protein Engineering Network Centers of Excellence, Biotechnology Laboratory and the Departments of ‡Chemical Engineering and §Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
Abstract
The surface diffusion rate of bacterial cellulases from Cellulomonas fimi on cellulose was quantified using fluorescence recovery after photobleaching analysis. Studies were performed on an exo-β-1–4-glycanase (Cex), an endo-β-1–4-glucanase (CenA), and their respective isolated cellulose-binding domains (CBDs). Although these cellulose-binding domains bind irreversibly to microcrystalline cellulose, greater than 70% of bound molecules are mobile on the cellulose surface. Surface diffusion rates are dependent on surface coverage and range from a low of 2 × 10−11 to a maximum of 1.2 × 10−10 cm2/s. The fraction of mobile molecules increases only slightly with increasing fractional surface coverage density. Results demonstrate that the packing of C. fimicellulases and their isolated binding domains onto the cellulose surface is a dynamic process. This suggests that the exclusion of potential CBD binding sites on the cellulose due to steric effects of neighboring bound CBDs may not fully explain the apparent negative cooperativity exhibited in CBD adsorption isotherms. Comparison with the kinetics of cellulase hydrolysis of crystalline substrate suggests that surface diffusion rates do not limit cellulase activity.
- Received February 20, 1997.
- Revision received June 2, 1997.
