Journal
CELLULOSE
Volume 18, Issue 3, Pages 749-758Publisher
SPRINGER
DOI: 10.1007/s10570-011-9506-2
Keywords
CLSM; Cellulose; Enzyme binding; kinetics; Transient model
Funding
- National Science Foundation [ECS-0335765]
- Department Of Energy [GO18084]
- New York State Foundation for Science, Technology and Innovation
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We report on studies of Thermobifida fusca cellulases Cel5A, Cel6B and Cel9A binding to pretreated wood particles using Confocal Laser Scanning Microscopy (CLSM). Hydro-thermal pretreated wood particles were immobilized on borosilicate substrates before fluorescently-labeled cellulase solutions at various concentrations were added. Time-lapse CLSM revealed that cellulases Cel5A, Cel6B and Cel9A quickly bound to certain areas of wood particles, slowly diffused into and adsorbed to less accessible areas, but showed little affinity for other areas of the wood. Cellulase-to-substrate association constants were estimated using a transient enzyme binding kinetics model, and were found to be in agreement with published values. In order to accurately account for the fluorescence signal of labeled enzyme mixed with wood autofluorescence, we also developed a spectral deconvolution method to separate signals from multiple fluorochromes.
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