Biomaterial Database

Reversibility and binding kinetics of Thermobifida fusca cellulases studied through fluorescence recovery after photobleaching microscopy. 2011

Jose M Moran-Mirabal, and Jacob C Bolewski, and Larry P Walker

Department of Biological and Environmental Engineering, Cornell University, Ithaca, NY 14853, USA. jmm248@cornell.edu

Cellulases are enzymes capable of depolymerizing cellulose. Understanding their interactions with cellulose can improve biomass saccharification and enzyme recycling in biofuel production. This paper presents a study on binding and binding reversibility of Thermobifida fusca cellulases Cel5A, Cel6B, and Cel9A bound onto Bacterial Microcrystalline Cellulose. Cellulase binding was assessed through fluorescence recovery after photobleaching (FRAP) at 23, 34, and 45 °C. It was found that cellulase binding is only partially reversible. For processive cellulases Cel6B and Cel9A, an increase in temperature resulted in a decrease of the fraction of cellulases reversibly bound, while for endocellulase Cel5A this fraction remained constant. Kinetic parameters were obtained by fitting the FRAP curves to a binding-dominated model. The unbinding rate constants obtained for all temperatures were highest for Cel5A and lowest for Cel9A. The results presented demonstrate the usefulness of FRAP to access the fast binding kinetics characteristic of cellulases operating at their optimal temperature.

UI	MeSH Term	Description	Entries
D007700	Kinetics	The rate dynamics in chemical or physical systems.
D008856	Microscopy, Fluorescence	Microscopy of specimens stained with fluorescent dye (usually fluorescein isothiocyanate) or of naturally fluorescent materials, which emit light when exposed to ultraviolet or blue light. Immunofluorescence microscopy utilizes antibodies that are labeled with fluorescent dye.	Fluorescence Microscopy,Immunofluorescence Microscopy,Microscopy, Immunofluorescence,Fluorescence Microscopies,Immunofluorescence Microscopies,Microscopies, Fluorescence,Microscopies, Immunofluorescence
D011485	Protein Binding	The process in which substances, either endogenous or exogenous, bind to proteins, peptides, enzymes, protein precursors, or allied compounds. Specific protein-binding measures are often used as assays in diagnostic assessments.	Plasma Protein Binding Capacity,Binding, Protein
D002480	Cellulase	An endocellulase with specificity for the hydrolysis of 1,4-beta-glucosidic linkages in CELLULOSE, lichenin, and cereal beta-glucans.	Endo-1,4-beta-Glucanase,Cellulysin,Endoglucanase,Endoglucanase A,Endoglucanase C,Endoglucanase E,Endoglucanase IV,Endoglucanase Y,beta-1,4-Glucan-4-Glucanohydrolase,Endo 1,4 beta Glucanase,beta 1,4 Glucan 4 Glucanohydrolase
D000192	Actinomycetales	An order of gram-positive, primarily aerobic BACTERIA that tend to form branching filaments.	Corynebacteriaceae,Coryneform Group
D013696	Temperature	The property of objects that determines the direction of heat flow when they are placed in direct thermal contact. The temperature is the energy of microscopic motions (vibrational and translational) of the particles of atoms.	Temperatures
D044602	Cellulases	A family of glycosidases that hydrolyse crystalline CELLULOSE into soluble sugar molecules. Within this family there are a variety of enzyme subtypes with differing substrate specificities that must work together to bring about complete cellulose hydrolysis. They are found in structures called CELLULOSOMES.	Laminarinase,Avicelase,Avicelase II,Cellulase Enzyme System,Cellulase Enzyme Systems,Endocellulase,Endocellulases,Endoglucanase I,Exo-cellulase,Exocellulase,Exocellulases,beta-Glucosidases,Enzyme System, Cellulase,Enzyme Systems, Cellulase,Exo cellulase,Systems, Cellulase Enzyme,beta Glucosidases
D036681	Fluorescence Recovery After Photobleaching	A method used to study the lateral movement of MEMBRANE PROTEINS and LIPIDS. A small area of a cell membrane is bleached by laser light and the amount of time necessary for unbleached fluorescent marker-tagged proteins to diffuse back into the bleached site is a measurement of the cell membrane's fluidity. The diffusion coefficient of a protein or lipid in the membrane can be calculated from the data. (From Segen, Current Med Talk, 1995).	Fluorescence Photobleaching Recovery,FRAP (Fluorescence Recovery After Photobleaching),FRAPs (Fluorescence Recovery After Photobleaching)