title: Hydrolysis of model cellulose films by cellulosomes: Extension of quartz crystal microbalance technique to multienzymatic complexes.
abstract: Bacterial cellulosomes contain highly efficient complexed cellulases and have been studied extensively for the production of lignocellulosic biofuels and bioproducts. A surface measurement technique, quartz crystal microbalance with dissipation (QCM-D), was extended for the investigation of real-time binding and hydrolysis of model cellulose surfaces from free fungal cellulases to the cellulosomes of Clostridium thermocellum (Ruminiclostridium thermocellum). In differentiating the activities of cell-free and cell-bound cellulosomes, greater than 68% of the cellulosomes in the crude cell broth were found to exist unattached to the cell across multiple growth stages. The initial hydrolysis rate of crude cell broth measured by QCM was greater than that of cell-free cellulosomes, but the corresponding frequency drop (a direct measure of the mass of enzyme adsorbed to the film) of crude cell broth was less than that of the cell-free cellulosomes, consistent with the underestimation of the cell mass adsorbed using QCM. Inhibition of hydrolysis by cellobiose (0-10g/L), which is similar for crude cell broth and cell-free cellulosomes, demonstrates the sensitivity of the QCM to environmental perturbations of multienzymatic complexes. QCM measurements using multienzymatic complexes may be used to screen and optimize hydrolysis conditions and to develop mechanistic, surface-based models of enzymatic cellulose deconstruction.
authors.affiliation: Department of Chemical and Materials Engineering, United States.
authors.forename: Shanshan
authors.initials: S
authors.lastname: Zhou
authors.affiliation: Department of Chemical and Materials Engineering, United States.
authors.forename: Hsin-Fen
authors.initials: HF
authors.lastname: Li
authors.affiliation: Department of Chemical and Materials Engineering, United States.
authors.forename: Ravinder
authors.initials: R
authors.lastname: Garlapalli
authors.affiliation: Department of Biosystems and Agricultural Engineering, United States.
authors.forename: Sue E
authors.initials: SE
authors.lastname: Nokes
authors.affiliation: Department of Animal and Food Sciences, University of Kentucky, Lexington, KY 40506, United States; USDA, Agricultural Research Service, Forage-Animal Production Research Unit; Lexington, KY 40546, United States.
authors.forename: Michael
authors.initials: M
authors.lastname: Flythe
authors.affiliation: Department of Chemical and Materials Engineering, United States.
authors.forename: Stephen E
authors.initials: SE
authors.lastname: Rankin
authors.affiliation: Department of Chemical and Materials Engineering, United States. Electronic address: bknutson@engr.uky.edu.
authors.forename: Barbara L
authors.initials: BL
authors.lastname: Knutson
mesh_terms: D002480:Cellulase / Q000378:metabolism; D002482:Cellulose / Q000737:chemistry / Q000378:metabolism*; D044603:Cellulosomes / Q000378:metabolism*; D048013:Acetivibrio thermocellus / Q000201:enzymology / Q000235:genetics; D005656:Fungal Proteins / Q000378:metabolism; D006868:Hydrolysis; D058525:Quartz Crystal Microbalance Techniques / Q000379:methods*
chemical_list: D005656:Fungal Proteins; D002482:Cellulose; D002480:Cellulase
keywords: Cellobiose inhibition; Cellulosomes; QCM-D; Solid substrate