title: Reversibility of substrate adsorption for the cellulases Cel7A, Cel6A, and Cel7B from Hypocrea jecorina.
abstract: Adsorption of cellulases on the cellulose surface is an integral part of the catalytic mechanism, and a detailed description of the adsorption process is therefore required for a fundamental understanding of this industrially important class of enzymes. However, the mode of adsorption has proven intricate, and several key questions remain open. Perhaps most notably it is not clear whether the adsorbed enzyme is in dynamic equilibrium with the free population or irreversibly associated with no or slow dissociation. To address this, we have systematically investigated adsorption reversibility for two cellobiohydrolases (Cel7A and Cel6A) and one endoglucanase (Cel7B) on four types of pure cellulose substrates. Specifically, we monitored dilution-induced release of adsorbed enzyme in samples that had previously been brought to a steady state (constant concentration of free enzyme). In simple dilution experiments (without centrifugation), the results consistently showed full reversibility. In contrast to this, resuspension of enzyme-substrate pellets separated by centrifugation showed extensive irreversibility. We conclude that these enzymes are in a dynamic equilibrium between free and adsorbed states but suggest that changes in the physical properties of cellulose caused by compaction of the pellet hampers subsequent release of adsorbed enzyme. This latter effect may be pertinent to both previous controversies in the literature on adsorption reversibility and the development of enzyme recycling protocols in the biomass industry.
authors.affiliation: Research Unit for Functional Biomaterials, NSM, Roskilde University , 1 Universitetsvej, Build. 18.1, DK-4000 Roskilde, Denmark.
authors.forename: Vanessa O A
authors.initials: VO
authors.lastname: Pellegrini
authors.forename: Nina
authors.initials: N
authors.lastname: Lei
authors.forename: Madhuri
authors.initials: M
authors.lastname: Kyasaram
authors.forename: Johan P
authors.initials: JP
authors.lastname: Olsen
authors.forename: Silke F
authors.initials: SF
authors.lastname: Badino
authors.forename: Michael S
authors.initials: MS
authors.lastname: Windahl
authors.forename: Francieli
authors.initials: F
authors.lastname: Colussi
authors.forename: Nicolaj
authors.initials: N
authors.lastname: Cruys-Bagger
authors.forename: Kim
authors.initials: K
authors.lastname: Borch
authors.forename: Peter
authors.initials: P
authors.lastname: Westh
mesh_terms: D000327:Adsorption; D044602:Cellulases / Q000737:chemistry*; D002482:Cellulose / Q000737:chemistry*; D005656:Fungal Proteins / Q000737:chemistry*; D035901:Hypocrea / Q000201:enzymology*
chemical_list: D005656:Fungal Proteins; D002482:Cellulose; D044602:Cellulases