Observing Thermobifida fusca cellulase binding to pretreated wood particles using time-lapse confocal laser scanning microscopy
- 354 Downloads
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.
KeywordsCLSM Cellulose Enzyme binding kinetics Transient model
Borafloat wafers were micro-patterned at the Cornell Nanoscale Science and Technology Facility, a member of the National Nanotechnology Infrastructure Network, which is supported by the National Science Foundation (Grant ECS-0335765). This research was funded by Department Of Energy Contract GO18084. This research made use of the Biofuel Research Laboratories at Cornell University, which was funded by the New York State Foundation for Science, Technology and Innovation.
- Browning, BL (ed) (1963) The chemistry of wood. Wiley, New YorkGoogle Scholar
- Ha T (2001) Single-molecule fluorescence resonance energy transfer. Methods Biochem Anal 25:78–86Google Scholar
- Johnson KA (2005) Enzyme kinetics: transient phase. In: Encyclopedia of life sciences. Wiley, New YorkGoogle Scholar
- Jung E, Lao G, Irwin D, Barr B, Benjamin A, Wilson D (1993) Dna-sequences and expression in Streptomyces lividans of an exoglucanase gene and an endoglucanase gene from Thermomonospora fusca. Appl Environ Microbiol 59(9):3032–3043Google Scholar
- Roughton FJW (1959) Diffusion and simultaneous chemical reaction velocity in haemoglobin solutions and red cell suspensions. Prog Biophys Biophys Chem 9:55–104Google Scholar