title: Multi-mode binding of Cellobiohydrolase Cel7A from Trichoderma reesei to cellulose.
abstract: Enzymatic hydrolysis of recalcitrant polysaccharides like cellulose takes place on the solid-liquid interface. Therefore the adsorption of enzymes to the solid surface is a pre-requisite for catalysis. Here we used enzymatic activity measurements with fluorescent model-substrate 4-methyl-umbelliferyl-β-D-lactoside for sensitive monitoring of the binding of cellobiohydrolase TrCel7A from Trichoderma reesei to bacterial cellulose (BC). The binding at low nanomolar free TrCel7A concentrations was exclusively active site mediated and was consistent with Langmuir's one binding site model with Kd and Amax values of 2.9 nM and 126 nmol/g BC, respectively. This is the strongest binding observed with non-complexed cellulases and apparently represents the productive binding of TrCel7A to cellulose chain ends on the hydrophobic face of BC microfibril. With increasing free TrCel7A concentrations the isotherm gradually deviated from the Langmuir's one binding site model. This was caused by the increasing contribution of lower affinity binding modes that included both active site mediated binding and non-productive binding with active site free from cellulose chain. The binding of TrCel7A to BC was found to be only partially reversible. Furthermore, the isotherm was dependent on the concentration of BC with more efficient binding observed at lower BC concentrations. The phenomenon can be ascribed to the BC concentration dependent aggregation of BC microfibrils with concomitant reduction of specific surface area.
authors.affiliation: Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Jalak
authors.affiliation: Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia.
authors.forename: Priit
authors.initials: P
authors.lastname: Väljamäe
mesh_terms: D020134:Catalytic Domain; D002482:Cellulose / Q000378:metabolism*; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000378:metabolism*; D011485:Protein Binding; D014242:Trichoderma / Q000201:enzymology*
chemical_list: D002482:Cellulose; D043366:Cellulose 1,4-beta-Cellobiosidase