SURFACE-DIFFUSION OF CELLULASES AND THEIR ISOLATED BINDING DOMAINS ONCELLULOSE

The surface diffusion rate of bacterial cellulases from Cellulomonas f imi on cellulose was quantified using fluorescence recovery after phot obleaching analysis. Studies were performed on an exo-beta-1-4-glycana se (Cex), an endo-beta-1-4-glucanase (CenA), and their respective isol ated cellulose-binding domains (CBDs). Although these cellulose-bindin g domains hind irreversibly to microcrystalline cellulose, greater tha n 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 x 10(-11) to a maximum of 1.2 x 10(-10) cm(2)/s. The fraction of mobile molecules increases only slightly with increasing fractional s urface coverage density. Results demonstrate that the packing of C. fi mi cellulases and their isolated binding domains onto the cellulose su rface is a dynamic process. This suggests that the exclusion of potent ial CBD binding sites on the cellulose due to steric effects of neighb oring bound CBDs may not fully explain the apparent negative cooperati vity exhibited in CBD adsorption isotherms. Comparison with the kineti cs of cellulase hydrolysis of crystalline substrate suggests that surf ace diffusion rates do not limit cellulase activity.