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Keywords:

  • adsorption;
  • atomic force microscopy;
  • cellulase;
  • cellulose morphology;
  • hydrolysis kinetics

Highly heterogeneous and usually weakly defined substrate morphologies complicate the study of enzymatic cellulose hydrolysis. The cellulose surface has a non-uniform shape in particular, with consequent impacts on cellulase adsorption and activity. We have therefore prepared a cellulosic model substrate which is shown by atomic force microscopy (AFM) to display a completely smooth surface, the residual squared mean roughness being 10 nm or lower, and applied it for kinetic analysis of cellulase action. The substrate consists of an amorphous cellulose matrix into which variably sized crystalline fibers are distributed in apparently irregular fashion. Its conversion into soluble sugars by Trichoderma sp. cellulase at 50 °C proceeded without apparent limitation up to 70% completion and was paralleled by a steady increase in cellulase adsorption to the cellulose. Individual cellulase components (CBH I, CBH II, EG) also showed strongly enhanced adsorption with progressing cellulose conversion, irrespective of their preference for degrading the amorphous or crystalline substrate parts as revealed by AFM. The specific activity of the adsorbed cellulases, however, decreased concomitantly. Cellulose surface morphologies evolving as a consequence of cellulase action were visualized by AFM. Three-dimensional surface degradation by the cellulases resulted in a large increase in cellulose surface area for enzyme adsorption. However, the decline in enzyme specific activity during conversion was caused by factors other than surface ablation and disruption. Based on kinetic evidence for enzymatic hydrolyses of the smooth-surface model substrate and microcrystalline cellulose (Avicel), we hypothesize that, due to gradual loss of productive dynamics in their interactions with the cellulose surface, individual cellulases get progressively confined to substrate parts where they are no longer optimally active. This eventually leads to an overall slow-down of hydrolysis.