title: In situ stability of substrate-associated cellulases studied by DSC.
abstract: This work shows that differential scanning calorimetry (DSC) can be used to monitor the stability of substrate-adsorbed cellulases during long-term hydrolysis of insoluble cellulose. Thermal transitions of adsorbed enzyme were measured regularly in subsets of a progressing hydrolysis, and the size of the transition peak was used as a gauge of the population of native enzyme. Analogous measurements were made for enzymes in pure buffer. Investigations of two cellobiohydrolases, Cel6A and Cel7A, from Trichoderma reesei, which is an anamorph of the fungus Hypocrea jerorina, showed that these enzymes were essentially stable at 25 °C. Thus, over a 53 h experiment, Cel6A lost less than 15% of the native population and Cel7A showed no detectable loss for either the free or substrate-adsorbed state. At higher temperatures we found significant losses in the native populations, and at the highest tested temperature (49 °C) about 80% Cel6A and 35% of Cel7A was lost after 53 h of hydrolysis. The data consistently showed that Cel7A was more long-term stable than Cel6A and that substrate-associated enzyme was less long-term stable than enzyme in pure buffer stored under otherwise equal conditions. There was no correlation between the intrinsic stability, specified by the transition temperature in the DSC, and the long-term stability derived from the peak area. The results are discussed with respect to the role of enzyme denaturation for the ubiquitous slowdown observed in the enzymatic hydrolysis of cellulose.
authors.affiliation: Research Unit for Functional Biomaterials, NSM, Roskilde University. 1 Universitetsvej , Build. 18.1, DK-4000 Roskilde Denmark.
authors.forename: Kadri
authors.initials: K
authors.lastname: Alasepp
authors.forename: Kim
authors.initials: K
authors.lastname: Borch
authors.forename: Nicolaj
authors.initials: N
authors.lastname: Cruys-Bagger
authors.forename: Silke
authors.initials: S
authors.lastname: Badino
authors.forename: Kenneth
authors.initials: K
authors.lastname: Jensen
authors.forename: Trine H
authors.initials: TH
authors.lastname: Sørensen
authors.forename: Michael S
authors.initials: MS
authors.lastname: Windahl
authors.forename: Peter
authors.initials: P
authors.lastname: Westh
mesh_terms: D002152:Calorimetry, Differential Scanning / Q000379:methods*; D044602:Cellulases / Q000737:chemistry* / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000378:metabolism; D004795:Enzyme Stability; D013696:Temperature; D014242:Trichoderma / Q000201:enzymology
chemical_list: D002482:Cellulose; D044602:Cellulases; D043366:Cellulose 1,4-beta-Cellobiosidase