Cellulose

, Volume 18, Issue 3, pp 749–758 | Cite as

Observing Thermobifida fusca cellulase binding to pretreated wood particles using time-lapse confocal laser scanning microscopy

  • Paul Zhu
  • Jose M. Moran-Mirabal
  • Jeremy S. Luterbacher
  • Larry P. Walker
  • Harold G. Craighead
Article

Abstract

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.

Keywords

CLSM Cellulose Enzyme binding kinetics Transient model 

Supplementary material

10570_2011_9506_MOESM1_ESM.pdf (387 kb)
PDF (388 KB)

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Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Paul Zhu
    • 1
  • Jose M. Moran-Mirabal
    • 2
  • Jeremy S. Luterbacher
    • 3
  • Larry P. Walker
    • 2
  • Harold G. Craighead
    • 1
  1. 1.Applied Engineering & PhysicsCornell UniversityIthacaUSA
  2. 2.Biological & Environmental EngineeringCornell UniversityIthacaUSA
  3. 3.Chemical & Biomolecular EngineeringCornell UniversityIthacaUSA

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